Antibodies that specifically bind to TL5

ABSTRACT

The present invention relates to antibodies and related molecules that specifically bind to TL5. Such antibodies have uses, for example, in the prevention and treatment of cancer as well as immune system diseases and disorders including autoimmune disease, rheumatoid arthritis, graft rejection, graft vs. host disease, and lymphadenopathy. The invention also relates to nucleic acid molecules encoding anti-TL5 antibodies, vectors and host cells containing these nucleic acids, and methods for producing the same. The present invention relates to methods and compositions for preventing, detecting, diagnosing, treating or ameliorating a disease or disorder, especially cancer as well as immune system diseases and disorders including autoimmune disease, rheumatoid arthritis, graft rejection, graft vs. host disease, and lymphadenopathy, comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that specifically bind to TL5.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT/US03/10956, filed Apr.10, 2003, which in turn claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 60/372,087, filed Apr. 15, 2002, each ofwhich applications is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to antibodies and related molecules thatspecifically bind to TL5. Such antibodies have uses, for example, in thediagnosis, prevention, and treatment of cancer as well as immune systemdiseases and disorders including autoimmune disease, rheumatoidarthritis, graft rejection, graft vs. host disease, and lymphadenopathy.The invention also relates to nucleic acid molecules encoding anti-TL5antibodies, vectors and host cells containing these nucleic acids, andmethods for producing the same. The present invention relates to methodsand compositions for preventing, detecting, diagnosing, treating orameliorating a disease or disorder, especially cancer as well as immunesystem diseases and disorders including autoimmune disease, rheumatoidarthritis, graft rejection, graft vs. host disease, and lymphadenopathy,comprising administering to an animal, preferably a human, an effectiveamount of one or more antibodies or fragments or variants thereof, orrelated molecules, that specifically bind to TL5.

BACKGROUND OF THE INVENTION

Human tumor necrosis factors α (TNFα) and β (TNFβ, or lymphotoxin) arerelated members of a broad class of polypeptide mediators, whichincludes the interferons, interleukins and growth factors, collectivelycalled cytokines (Beutler, B. and Cerami, A., Annu. Rev. Immunol.7:625-655 (1989)).

Tumor necrosis factor (TNFα and TNFβ) was originally discovered as aresult of its anti-tumor activity, however, now it is recognized as apleiotropic cytokine capable of numerous biological activities includingapoptosis of some transformed cell lines, mediation of cell activationand proliferation and also as playing important roles in immuneregulation and inflammation.

To date, known members of the TNF-ligand superfamily include TNFα, TNFβ(lymphotoxin-α, LTβ, OX40L, Fas ligand, CD30L, CD27L, CD40L and 4-IBBL.The ligands of the TNF ligand superfamily are acidic, TNF-like moleculeswith approximately 20% sequence homology in the extracellular domains(range, 12%-36%) and exist mainly as membrane-bound forms with thebiologically active form being a trimeric/multimeric complex. Solubleforms of the TNF ligand superfamily have only been identified so far forTNF, LTα, and Fas ligand (for a general review, see Gruss, H. and Dower,S. K., Blood, 85(12):3378-3404 (1995)), which is hereby incorporated byreference in its entirety.

These proteins are involved in regulation of cell proliferation,activation, and differentiation, including control of cell survival ordeath by apoptosis or cytotoxicity (Armitage, R. J., Curr. Opin.Immunol. 6:407 (1994) and Smith, C. A., Cell 75:959 (1994)).

Mammalian development is dependent on both the proliferation anddifferentiation of cells as well as programmed cell death which occursthrough apoptosis (Walker et al., Methods Achiev. Exp. Pathol. 13:18(1988). Apoptosis plays a critical role in the destruction of immunethymocytes that recognize self antigens. Failure of this normalelimination process may play a role in autoimmune diseases (Gammon etal., Immunology Today 12:193 (1991)).

Itoh et al. (Cell 66:233 (1991)) described a cell surface antigen,Fas/CD95 that mediates apoptosis and is involved in clonal deletion of Tcells. Fas is expressed in activated T cells, B-cells, neutrophils andin thymus, liver, heart and lung and ovary in adult mice(Watanabe-Fukunaga et al., J. Immunology. 148:1274 (1992)). Inexperiments where a monoclonal Ab to Fas is cross-linked to Fas,apoptosis is induced (Yonehara et al.,J. Exp. Med. 169:1747 (1989);Trauth et al., Science 245:301 (1989)). In addition, there is an examplewhere binding of a monoclonal Ab to Fas may stimulate T cells undercertain conditions (Alderson et al., J. Exp. Med. 178:2231 (1993)).

Fas antigen is a cell surface protein of relative MW of 45 Kd. Bothhuman and murine genes for Fas have been cloned by Watanabe-Fukunaga etal., (J. Immunol. 148:1274 (1992)) and Itoh et al. (Cell 66:233 (1991)).The proteins encoded by these genes are both transmembrane proteins withstructural homology to the Nerve Growth Factor/Tumor Necrosis Factorreceptor superfamily, which includes two TNF receptors, the low affinityNerve Growth Factor receptor and the LTβ receptor CD40, CD27, CD30, andOX40.

Recently the Fas ligand has been described (Suda et al., Cell 75:1169(1993)). The amino acid sequence indicates that Fas ligand is a type IItransmembrane protein belonging to the TNF family. Fas ligand isexpressed in splenocytes and thymocytes. The purified Fas ligand has aMW of 40 kd.

Recently, it has been demonstrated that Fas/Fas ligand interactions arerequired for apoptosis following the activation of T cells (Ju et al.,Nature 373:444 (1995); Brunner et al., Nature 373:441 (1995)).Activation of T cells induces both proteins on the cell surface.Subsequent interaction between the ligand and receptor results inapoptosis of the cells. This supports the possible regulatory role forapoptosis induced by Fas/Fas ligand interaction during normal immuneresponses.

The polypeptide of the present invention has been identified as a novelmember of the TNF ligand super-family based on structural and biologicalsimilarities.

The effects of TNF family ligands and TNF family receptors are variedand influence numerous functions, both normal and abnormal, in thebiological processes of the mammalian system. There is a clear need,therefore, for identification and characterization of compositions, suchas antibodies, that influence the biological activity of TNF receptors,both normally and in disease states. In particular, there is a need toisolate and characterize antibodies that modulate the biologicalactivities of TL5. Such antibodies will be useful for, example, in thetreatment of autoimmune disease, graft versus host disease, rheumatoidarthritis, lymphadenopathy, and cancer.

SUMMARY OF THE INVENTION

The present invention encompasses antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof) that specifically bind to a TL5 polypeptide orpolypeptide fragment or variant of TL5. In particular, the inventionencompasses antibodies (including molecules comprising, or alternativelyconsisting of, antibody fragments or variants thereof) that specificallybind to a TL5 polypeptide (SEQ ID NO:2) or polypeptide fragment orvariant of human TL5 such as that of SEQ ID NO:4.

The present invention relates to methods and compositions forpreventing, treating or ameliorating a disease or disorder comprisingadministering to an animal, preferably a human, an effective amount ofone or more antibodies or fragments or variants thereof, or relatedmolecules, that specifically bind to TL5 or a fragment or variantthereof. In specific embodiments, the present invention relates tomethods and compositions for preventing, treating or ameliorating adisease or disorder associated with TL5 function or aberrant TL5expression, comprising administering to an animal, preferably a human,an effective amount of one or more antibodies or fragments or variantsthereof, or related molecules, that specifically bind to TL5 or afragment or variant thereof.

In highly preferred embodiments, the present invention encompassesmethods for using the antibodies of the present invention to treat,prevent, diagnose and/or prognose a disease or disorder of the immunesystem. In preferred embodiments, the present invention encompassesmethods for using antibodies of the invention to treat, prevent,diagnose and/or prognose a disease or disorder of the immune system.

In other preferred embodiments, the invention encompasses methods forusing the antibodies of the invention to treat, prevent, diagnose and/orprognose a disease or disorder associated with aberrant T cellactivation. In specific embodiments, the invention encompasses methodsfor using the antibodies of the present invention to treat, prevent,diagnose and/or prognose a disease or disorder associated with aberrantCD8⁺ T cell activation. In further specific embodiments, the inventionencompasses methods for using the polynucleotides, polypeptides orantibodies of the present invention to treat, prevent, diagnose and/orprognose a disease or disorder associated with aberrant Th1 celldifferentiation and/or function.

In highly preferred embodiments, the present invention relates toantibody-based methods and compositions for preventing, treating orameliorating cancers and other hyperproliferative disorders (e.g.,leukemia, carcinoma, and lymphoma). In other highly preferredembodiments, the present invention relates to antibody-based methods andcompositions for preventing, treating or ameliorating immune disorders(e.g., autoimmune disorders, graft-versus-host disease, lupus,rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto'sdisease, immunodeficiency syndrome, and Graves' disease). In additionalhighly preferred embodiments, the present invention relates toantibody-based methods and compositions for preventing, treating orameliorating inflammatory disorders (e.g., lymphadenopathy, asthma,allergic disorders, anaphylaxis, adult respiratory distress syndrome,and Crohn's disease).

In other highly preferred embodiments, the present invention relates toantibody-based methods and compositions for treating, preventing,diagnosing, and/or prognosing septic shock, inflammation, cerebralmalaria, activation of the HIV virus, graft-host rejection,immunodeficiency, bone resorption, and cachexia (wasting ormalnutrition). In a preferred embodiment, anti-TL5 antibodies are usedto treat, prevent, diagnose, and/or prognose graft versus host disease.

In one embodiment, antibodies of the invention are used to prevent,diagnose, treat or ameliorate a disease or disorder associated withaberrant T cell (e.g., Th1 or CD8+ T cells) activity, for example,aberrant T cell proliferation, differentiation, or effector function. Ina specific embodiment, antibodies of the invention are used to prevent,diagnose, treat or ameliorate a disease or disorder associated withincreased or excess T cell (e.g., Th1 or CD8+ T cells) activity, forexample, increased or excess T cell proliferation, differentiation, oreffector function.

In a further aspect of the invention, antibodies of the presentinvention may be used to treat rheumatoid arthritis (RA).

Another embodiment of the present invention includes the use of theantibodies of the invention as a diagnostic tool to monitor theexpression of TL5.

The present invention encompasses single chain Fv's (scFvs) thatspecifically bind TL5 polypeptides (e.g., SEQ ID NOs:5-7). Thus, theinvention encompasses these scFvs, listed in Table 1. In addition theinvention encompasses cell lines engineered to express antibodiescorresponding to these scFvs which have been deposited with the AmericanType Culture Collection (“ATCC”) as of the dates listed in Table 1 andgiven the ATCC Deposit Numbers identified in Table 1 The ATCC is locatedat 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCCdeposit was made pursuant to the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for Purposesof Patent Procedure.

Further, the present invention encompasses the polynucleotides encodingthe scFvs, as well as the amino acid sequences encoding the scFvs.Molecules comprising, or alternatively consisting of, fragments orvariants of these scFvs (e.g., VH domains, VH CDRs, VL domains, or VLCDRs having an amino acid sequence of any one of the correspondingportion of the scFvs referred to in Table 1), that specifically bind toTL5 or fragments or variants thereof are also encompassed by theinvention, as are nucleic acid molecules that encode these antibodiesand/or molecules. In highly preferred embodiments, the present inventionencompasses antibodies, or fragments or variants thereof, that bind tothe extracellular regions/domains of one or more TL5 polypeptides orfragments and variants thereof.

The present invention also anti-TL5 antibodies which are coupled to adetectable label, such as an enzyme, a fluorescent label, a luminescentlabel, or a bioluminescent label. The present invention also providesanti-TL5 antibodies which are coupled to a therapeutic or cytotoxicagent. The present invention also provides anti-TL5 polypeptides whichare coupled to a radioactive material.

The present invention also provides antibodies that specifically bindone or more TL5 polypeptides and that act as either TL5 agonists or TL5antagonists. In specific embodiments, the antibodies of the inventioninhibit TL5 binding to a TL5 receptor (e.g., TR6 (described in WO98/30694, WO2000/52028, WO2002/18622, and SEQ ID NO:49), LTI3R (See,e.g. GenBank™ Accession Numbers L04270 and P36941 and SEQ ID NO:47), andTR2 (described in WO 96/34095, WO98/18824, WO00/56405 and SEQ ID NO:48).Each of the publications and patents cited above is hereby incorporatedby reference in their entireties.

In specific embodiments, the antibodies of the invention inhibitproliferation of cells that express a TL5 receptor (e.g., TR2, TR6, orLTβR). In specific embodiments, the antibodies of the invention inhibitdifferentiation of cells (e.g., Th1 and CD8⁺ T cells) that express a TL5receptor (e.g., TR2, TR6, or LTβR). In other embodiments, the inventionprovides antibodies that inhibit T cell (e.g., Th1 and CD8⁺) secretionof IFN-•. In specific embodiments, the antibodies of the inventioninhibit apoptosis of TL5 expressing cells (e.g., T cells).

The present invention also provides antibodies that stimulateproliferation of cells that express a TL5 receptor (e.g., TR2, TR6, orLTβR). The present invention also provides antibodies that stimulatedifferentiation of cells (e.g., the differentiation of T cells into Th1or Tct11 and the differentiation and maturation of CD8⁺ T cells) thatexpress a TL5 receptor (e.g., TR2, TR6, or LTβR). In other embodiments,the invention provides antibodies that stimulate T cell (e.g., thedifferentiation of T cells into Th1 or Tct11 and the differentiation andmaturation of CD8⁺) secretion of IFN-•. In specific embodiments, theantibodies of the invention stimulate apoptosis of TL5 receptorexpressing cells (e.g., T cells).

In further embodiments, the antibodies of the invention have adissociation constant (K_(D)) of 10⁻⁷ M or less. In preferredembodiments, the antibodies of the invention have a dissociationconstant (K_(D)) of 10⁻⁹ M or less.

In further embodiments, antibodies of the invention have an off rate(k_(off)) of 10⁻³/sec or less. In preferred embodiments, antibodies ofthe invention have an off rate (k_(off)) of 10⁻⁴/sec or less. In otherpreferred embodiments, antibodies of the invention have an off rate(k_(off)) of 10⁻⁵/sec or less.

The present invention also provides panels of antibodies (includingmolecules comprising, or alternatively consisting of, antibody fragmentsor variants) wherein the panel members correspond to one, two, three,four, five, ten, fifteen, twenty, or more different antibodies of theinvention (e.g., whole antibodies, Fabs, F(ab′)₂ fragments, Fdfragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id)antibodies, and scFvs). The present invention further provides mixturesof antibodies, wherein the mixture corresponds to one, two, three, four,five, ten, fifteen, twenty, or more different antibodies of theinvention (e.g., whole antibodies, Fabs, F(ab′)₂ fragments, Fdfragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id)antibodies, and scFvs)). The present invention also provides forcompositions comprising, or alternatively consisting of, one, two,three, four, five, ten, fifteen, twenty, or more antibodies of thepresent invention (including molecules comprising, or alternativelyconsisting of, antibody fragments or variants thereof). A composition ofthe invention may comprise, or alternatively consist of, one, two,three, four, five, ten, fifteen, twenty, or more amino acid sequences ofone or more antibodies or fragments or variants thereof. Alternatively,a composition of the invention may comprise, or alternatively consistof, nucleic acid molecules encoding one or more antibodies of theinvention.

The present invention also provides for fusion proteins comprising anantibody (including molecules comprising, or alternatively consistingof, antibody fragments or variants thereof) of the invention, and aheterologous polypeptide (i.e., a polypeptide unrelated to an antibodyor antibody domain). Nucleic acid molecules encoding these fusionproteins are also encompassed by the invention. A composition of thepresent invention may comprise, or alternatively consist of, one, two,three, four, five, ten, fifteen, twenty or more fusion proteins of theinvention. Alternatively, a composition of the invention may comprise,or alternatively consist of, nucleic acid molecules encoding one, two,three, four, five, ten, fifteen, twenty or more fusion proteins of theinvention.

The present invention also provides for a nucleic acid molecule(s),generally isolated, encoding an antibody (including molecules, such asscFvs, VH domains, or VL domains, that comprise, or alternativelyconsist of, an antibody fragment or variant thereof) of the invention.The present invention also provides a host cell transformed with anucleic acid molecule of the invention and progeny thereof. The presentinvention also provides a method for the production of an antibody(including a molecule comprising, or alternatively consisting of, anantibody fragment or variant thereof) of the invention. The presentinvention further provides a method of expressing an antibody (includinga molecule comprising, or alternatively consisting of, an antibodyfragment or variant thereof) of the invention from a nucleic acidmolecule. These and other aspects of the invention are described infurther detail below.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “antibody,” as used herein, refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that specifically bindsan antigen. As such, the term antibody encompasses not only wholeantibody molecules, but also antibody multimers and antibody fragmentsas well as variants (including derivatives) of antibodies, antibodymultimers and antibody fragments. Examples of molecules which aredescribed by the term “antibody” herein include, but are not limited to:single chain Fvs (scFvs), Fab fragments, Fab′ fragments, F(ab′)₂,disulfide linked Fvs (sdFvs), Fvs, and fragments comprising oralternatively consisting of, either a VL or a VH domain. The term“single chain Fv” or “scFv” as used herein refers to a polypeptidecomprising a VH domain of antibody linked to a VL domain of an antibody.Antibodies that specifically bind to TL5 may have cross-reactivity withother antigens. Preferably, antibodies that specifically bind to TL5 donot cross-react with other antigens (e.g., other members of the TumorNecrosis Factor superfamily). Antibodies that specifically bind to TL5can be identified, for example, by immunoassays or other techniquesknown to those of skill in the art.

Antibodies of the invention include, but are not limited to, monoclonal,multispecific, human or chimeric antibodies, single chain antibodies,Fab fragments, F(ab′) fragments, anti-idiotypic (anti-Id) antibodies(including, e.g., anti-Id antibodies to antibodies of the invention),intracellularly-made antibodies (i.e., intrabodies), and epitope-bindingfragments of any of the above. The immunoglobulin molecules of theinvention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY),class (e.g. IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂) or subclass ofimmunoglobulin molecule. Preferably, an antibody of the inventioncomprises, or alternatively consists of, a VH domain, VH CDR, VL domain,or VL CDR having an amino acid sequence of any one of those referred toin Table 1, or a fragment or variant thereof. In a preferred embodiment,the immunoglobulin is an IgG1 isotype. In another preferred embodiment,the immunoglobulin is an IgG4 isotype. Immunoglobulins may have both aheavy and light chain. An array of IgG, IgE, IgM, IgD, IgA, and IgYheavy chains may be paired with a light chain of the kappa or lambdaforms.

Antibodies of the invention may also include multimeric forms ofantibodies. For example, antibodies of the invention may take the formof antibody dimers, trimers, or higher-order multimers of monomericimmunoglobulin molecules. Dimers of whole immunoglobulin molecules or ofF(ab′)2 fragments are tetravalent, whereas dimers of Fab fragments orscFv molecules are bivalent. Individual monomers within an antibodymultimer may be identical or different, i.e., they may be heteromeric orhomomeric antibody multimers. For example, individual antibodies withina multimer may have the same or different binding specificities.

Multimerization of antibodies may be accomplished through naturalaggregation of antibodies or through chemical or recombinant linkingtechniques known in the art. For example, some percentage of purifiedantibody preparations (e.g., purified IgG1 molecules) spontaneously formprotein aggregates containing antibody homodimers, and otherhigher-order antibody multimers. Alternatively, antibody homodimers maybe formed through chemical linkage techniques known in the art. Forexample, heterobifunctional crosslinking agents including, but notlimited to, SMCC [succinimidyl4-(maleimidomethyl)cyclohexane-1-carboxylate] and SATA [N-succinimidylS-acethylthio-acetate] (available, for example, from PierceBiotechnology, Inc. (Rockford, Ill.)) can be used to form antibodymultimers. An exemplary protocol for the formation of antibodyhomodimers is given in Ghetie et al., Proceedings of the NationalAcademy of Sciences USA (1997) 94:7509-7514, which is herebyincorporated by reference in its entirety. Antibody homodimers can beconverted to Fab′2 homodimers through digestion with pepsin. Another wayto form antibody homodimers is through the use of the autophilic T15peptide described in Zhao and Kohler, The Journal of Immunology (2002)25:396-404, which is hereby incorporated by reference in its entirety.

Alternatively, antibodies can be made to multimerize through recombinantDNA techniques. IgM and IgA naturally form antibody multimers throughthe interaction with the J chain polypeptide. Non-IgA or non-IgMmolecules, such as IgG molecules, can be engineered to contain the Jchain interaction domain of IgA or IgM, thereby conferring the abilityto form higher order multimers on the non-IgA or non-IgM molecules.(see, for example, Chintalacharuvu et al., (2001) Clinical Immunology101:21-31. and Frigerio et al., (2000) Plant Physiology 123:1483-94.,both of which are hereby incorporated by reference in their entireties.)ScFv dimers can also be formed through recombinant techniques known inthe art; an example of the construction of scFv dimers is given in Goelet al., (2000) Cancer Research 60:6964-6971 which is hereby incorporatedby reference in its entirety. Antibody multimers may be purified usingany suitable method known in the art, including, but not limited to,size exclusion chromatography.

By “isolated antibody” is intended an antibody removed from its nativeenvironment. Thus, for example, an antibody produced by, purified fromand/or contained within a hybridoma and/or a recombinant host cell isconsidered isolated for purposes of the present invention.

Unless otherwise defined in the specification, specific binding orimmunospecifc binding by an anti-TL5 antibody means that the anti-TL5antibody binds TL5 but does not significantly bind to (i.e., cross reactwith) proteins other than TL5, such as other proteins in the same familyof proteins). An antibody that binds TL5 protein and does notcross-react with other proteins is not necessarily an antibody that doesnot bind said other proteins in all conditions; rather, the TL5-specificantibody of the invention preferentially binds TL5 compared to itsability to bind said other proteins such that it will be suitable foruse in at least one type of assay or treatment, i.e., give lowbackground levels or result in no unreasonable adverse effects intreatment. It is well known that the portion of a protein bound by anantibody is known as the epitope. An epitope may either be linear (i.e.,comprised of sequential amino acids residues in a protein sequences) orconformational (i.e., comprised of one or more amino acid residues thatare not contiguous in the primary structure of the protein but that arebrought together by the secondary, tertiary or quaternary structure of aprotein). Given that TL5-specific antibodies bind to epitopes of TL5, anantibody that specifically binds TL5 may or may not bind fragments ofTL5 and/or variants of TL5 (e.g., proteins that are at least 90%identical to TL5) depending on the presence or absence of the epitopebound by a given TL5-specific antibody in the TL5 fragment or variant.Likewise, TL5-specific antibodies of the invention may bind speciesorthologues of TL5 (including fragments thereof) depending on thepresence or absence of the epitope recognized by the antibody in theorthologue. Additionally, TL5-specific antibodies of the invention maybind modified forms of TL5, for example, TL5 fusion proteins. In such acase when antibodies of the invention bind TL5 fusion proteins, theantibody must make binding contact with the TL5 moiety of the fusionprotein in order for the binding to be specific. Antibodies thatspecifically bind to TL5 can be identified, for example, by immunoassaysor other techniques known to those of skill in the art, e.g., theimmunoassays described in the Examples below.

The term “variant” as used herein refers to a polypeptide that possessesa similar or identical amino acid sequence as a TL5 polypeptide, afragment of a TL5 polypeptide, an anti-TL5 antibody or antibody fragmentthereof. A variant having a similar amino acid sequence refers to apolypeptide that satisfies at least one of the following: (a) apolypeptide comprising, or alternatively consisting of, an amino acidsequence that is at least 30%, at least 35%, at least 40%, at least 45%,at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95% or atleast 99% identical to the amino acid sequence of a TL5 polypeptide, afragment thereof, an anti-TL5 antibody or antibody fragment thereof(including a VH domain, VHCDR, VL domain, or VLCDR having an amino acidsequence of any one or more scFvs referred to in Table 1) describedherein; (b) a polypeptide encoded by a nucleotide sequence, thecomplementary sequence of which hybridizes under stringent conditions toa nucleotide sequence encoding a TL5 polypeptide (e.g., SEQ ID NO:2), afragment of a TL5 polypeptide, an anti-TL5 antibody or antibody fragmentthereof (including a VH domain, VHCDR, VL domain, or VLCDR having anamino acid sequence of any one of those referred to in Table 1),described herein, of at least 5 amino acid residues, at least 10 aminoacid residues, at least 15 amino acid residues, at least 20 amino acidresidues, at least 25 amino acid residues, at least 30 amino acidresidues, at least 40 amino acid residues, at least 50 amino acidresidues, at least 60 amino residues, at least 70 amino acid residues,at least 80 amino acid residues, at least 90 amino acid residues, atleast 100 amino acid residues, at least 125 amino acid residues, or atleast 150 amino acid residues; and (c) a polypeptide encoded by anucleotide sequence that is at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% or at least 99%, identical to the nucleotide sequence encodinga TL5 polypeptide, a fragment of a TL5 polypeptide, an anti-TL5 antibodyor antibody fragment thereof (including a VH domain, VHCDR, VL domain,or VLCDR having an amino acid sequence of any one or more scFvs referredto in Table 1), described herein. A polypeptide with similar structureto a TL5 polypeptide, a fragment of a TL5 polypeptide, an anti-TL5antibody or antibody fragment thereof, described herein refers to apolypeptide that has a similar secondary, tertiary or quaternarystructure of a TL5 polypeptide, a fragment of a TL5 polypeptide, ananti-TL5 antibody, or antibody fragment thereof, described herein. Thestructure of a polypeptide can determined by methods known to thoseskilled in the art, including but not limited to, X-ray crystallography,nuclear magnetic resonance, and crystallographic electronmicroscopy._Preferably, a variant TL5 polypeptide, a variant fragment ofa TL5 polypeptide, or a variant anti-TL5 antibody and/or antibodyfragment possesses similar or identical function and/or structure as thereference TL5 polypeptide, the reference fragment of a TL5 polypeptide,or the reference anti-TL5 antibody and/or antibody fragment,respectively.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoacid or nucleic acid sequence). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide at the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=numberof identical overlapping positions/total number of positions×100%). Inone embodiment, the two sequences are the same length.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm known to those of skill inthe art. An example of a mathematical algorithm for comparing twosequences is the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci.USA 87:2264-2268(1990), modified as in Karlin and Altschul Proc. Natl.Acad. Sci. USA 90:5873-5877(1993). The BLASTn and BLASTx programs ofAltschul, et al. J. Mol. Biol. 215:403-410(1990) have incorporated suchan algorithm. BLAST nucleotide searches can be performed with the BLASTnprogram (score=100, wordlength=12) to obtain nucleotide sequenceshomologous to a nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the BLASTx program (score=50,wordlength=3) to obtain amino acid sequences homologous to a proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.Nucleic Acids Res. 25:3589-3402(1997). Alternatively, PSI-BLAST can beused to perform an iterated search which detects distant relationshipsbetween molecules (Id.). When utilizing BLAST, Gapped BLAST, andPSI-BLAST programs, the default parameters of the respective programs(e.g., BLASTx and BLASTn) can be used.

Another example of a mathematical algorithm utilized for the comparisonof sequences is the algorithm of Myers and Miller, CABIOS (1989). TheALIGN program (version 2.0) which is part of the GCG sequence alignmentsoftware package has incorporated such an algorithm. Other algorithmsfor sequence analysis known in the art include ADVANCE and ADAM asdescribed in Torellis and Robotti Comput. Appl. Biosci., 10:3-5(1994);and FASTA described in Pearson and Lipman Proc. Natl. Acad. Sci.85:2444-8(1988). Within FASTA, ktup is a control option that sets thesensitivity and speed of the search.

The term “derivative” as used herein, refers to a variant polypeptide ofthe invention that comprises, or alternatively consists of, an aminoacid sequence of a TL5 polypeptide, a fragment of a TL5 polypeptide, oran antibody of the invention that specifically binds to a TL5polypeptide, which has been altered by the introduction of amino acidresidue substitutions, deletions or additions. The term “derivative” asused herein also refers to a TL5 polypeptide, a fragment of a TL5polypeptide, or an antibody that specifically binds to a TL5 polypeptidewhich has been modified, e.g., by the covalent attachment of any type ofmolecule to the polypeptide. For example, but not by way of limitation,a TL5 polypeptide, a fragment of a TL5 polypeptide, or an anti-TL5antibody, may be modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. A derivative of a TL5 polypeptide, afragment of a TL5 polypeptide, or an anti-TL5 antibody, may be modifiedby chemical modifications using techniques known to those of skill inthe art, including, but not limited to, specific chemical cleavage,acetylation, formylation, metabolic synthesis of tunicamycin, etc.Further, a derivative of a TL5 polypeptide, a fragment of a TL5polypeptide, or an anti-TL5 antibody, may contain one or morenon-classical amino acids. A polypeptide derivative possesses a similaror identical function as a TL5 polypeptide, a fragment of a TL5polypeptide, or an anti-TL5 antibody, described herein.

The term “fragment” as used herein refers to a polypeptide comprising anamino acid sequence of at least 5 amino acid residues, at least 10 aminoacid residues, at least 15 amino acid residues, at least 20 amino acidresidues, at least 25 amino acid residues, at least 30 amino acidresidues, at least 35 amino acid residues, at least 40 amino acidresidues, at least 45 amino acid residues, at least 50 amino acidresidues, at least 60 amino residues, at least 70 amino acid residues,at least 80 amino acid residues, at least 90 amino acid residues, atleast 100 amino acid residues, at least 125 amino acid residues, atleast 150 amino acid residues, at least 175 amino acid residues, or atleast 200 amino acid residues, of the amino acid sequence of TL5, or ananti-TL5 antibody (including molecules such as scFv's, that comprise, oralternatively consist of, antibody fragments or variants thereof) thatspecifically binds to TL5.

The term “host cell” as used herein refers to the particular subjectcell transfected with a nucleic acid molecule and the progeny orpotential progeny of such a cell. Progeny may not be identical to theparent cell transfected with the nucleic acid molecule due to mutationsor environmental influences that may occur in succeeding generations orintegration of the nucleic acid molecule into the host cell genome.

Antibody Structure

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Humanlight chains are classified as kappa and lambda light chains. Heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, lgG, IgA, and IgE, respectively. Seegenerally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. RavenPress, N.Y. (1989)) (incorporated by reference in its entirety for allpurposes). The variable regions of each light/heavy chain pair form theantibody binding site.

Thus, an intact IgG antibody has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame.

The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hyper variable regions,also called complementarity determining regions or CDRs. The CDRs fromthe heavy and the ligt chains of each pair are aligned by the frameworkregions, enabling binding to a specific epitope. From N-terminal toC-terminal, both light and heavy chains comprise the domains FR1, CDR1,FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to eachdomain is in accordance with the definitions of Kabat Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J Mol. Biol.196:901-917-(1987); Chothia et al. Nature 342:878-883 (1989).

A bispecific or bifunctional antibody is an artificial hybrid antibodyhaving two different heavy/light chain pairs and two different bindingsites. Bispecific antibodies can be produced by a variety of methodsincluding fusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelnyet al. J Immunol. 148:1547 1553 (1992). In addition, bispecificantibodies may be formed as “diabodies” (Holliger et al. “‘Diabodies’:small bivalent and bispecific antibody fragments” PNAS USA 90:6444-6448(1993)) or “Janusins” (Traunecker et al. “Bispecific single chainmolecules (Janusins) target cytotoxic lymphocytes on HIV infected cells”EMBO J 10:3655-3659 (1991) and Traunecker et al. “Janusin: new moleculardesign for bispecific reagents” Int. J. Cancer Suppl. 7:51-52 (1992)).

Production of bispecific antibodies can be a relatively labor intensiveprocess compared with production of conventional antibodies and yieldsand degree of purity are generally lower for bispecific antibodies.Bispecific antibodies do not exist in the form of fragments having asingle binding site (e.g., Fab, Fab′, and Fv).

Anti-TL5 Antibodies

Using phage display technology, single chain antibody molecules(“scFvs”) have been identified that specifically bind to TL5 (orfragments or variants thereof). Molecules comprising, or alternativelyconsisting of, fragments or variants of these scFvs (e.g., including VHdomains, VH CDRs, VL domains, or VL CDRs having an amino acid sequenceof any one of those referred to in Table 1), that specifically bind toTL5 (or fragments or variants thereof) are also encompassed by theinvention, as are nucleic acid molecules that encode these scFvs, and/ormolecules.

In particular, the invention relates to scFvs comprising, oralternatively consisting of the amino acid of SEQ ID NOs: 5-7, referredto in Table 1 below. Molecules comprising, or alternatively consistingof, fragments or variants (e.g., including VH domains, VH CDRs, VLdomains, or VL CDRs identified in Table 1) of the scFvs referred to inTable 1, that specifically bind to TL5 are also encompassed by theinvention, as are nucleic acid molecules that encode these scFvs, and/ormolecules (e.g., SEQ ID NOs:8-10).

ScFvs corresponding to SEQ ID NOs:5-7 were selected for their abilitybind TL5 polypeptide.

The present invention provides antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof) that specifically bind to a polypeptide or apolypeptide fragment of TL5. In particular, the invention providesantibodies corresponding to the scFvs referred to in Table 1, such scFvsmay routinely be “converted” to immunoglobulin molecules by inserting,for example, the nucleotide sequences encoding the VH and/or VL domainsof the scFv into an expression vector containing the constant domainsequences and engineered to direct the expression of the immunoglobulinmolecule, as described in more detail in Example 1 below.

Cell lines that express IgG1 antibodies that comprise the VH and VLdomains of scFvs of the invention have been deposited with the AmericanType Culture Collection (“ATCC”) on the dates listed in Table 1 andgiven the ATCC Deposit Numbers identified in Table 1. The ATCC islocated at 10801 University Boulevard, Manassas, Va. 20110-2209, USA.The ATCC deposit was made pursuant to the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forPurposes of Patent Procedure.

Accordingly, in one embodiment, the invention provides antibodies thatcomprise the VH and VL domains of scFvs of the invention. In oneembodiment, the invention provides antibodies that comprise the VH andVL domains of the L006C05 scFv. In one embodiment, the inventionprovides antibodies that comprise the VH and VL domains of the L003B01scFv. In one embodiment, the invention provides antibodies that comprisethe VH and VL domains of the L044F07 scFv.

In a preferred embodiment, an antibody of the invention is the antibodyexpressed by cell line X1 (See Table 1).

In a preferred embodiment, an antibody of the invention is the antibodyexpressed by cell line X2 (See Table 1).

In a preferred embodiment, an antibody of the invention is the antibodyexpressed by cell line X3 (See Table 1).

TABLE 1 scFvs that Specifically bind to TL5 scFv scFv protein DNA AAsAAs of AAs of AAs of AAs of AAs of AAs of AAs of Cell Line ATCC ATCC SEQID SEQ ID of VH VH VH VH VL VL VL VL Expressing Deposit Deposit ScFv NO:NO: Domain CDR1 CDR2 CDR3 Domain CDR1 CDR2 CDR3 antibody Number DateL006C05 5 8 1-119 31-35 50-66 99-108 135-245 157-170 186-192 225-234 X1Y1 L003B01 6 9 1-122 31-35 50-66 99-111 138-248 160-173 189-195 228-237X2 Y2 L044F07 7 10 1-119 31-35 50-66 99-108 135-242 156-166 182-188221-231 X3 Y3

The present invention encompasses antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof) that specifically bind to a TL5 polypeptide or afragment, variant, or fusion protein thereof. A TL5 polypeptideincludes, but is not limited to, TL5 (SEQ ID NO:2) or the polypeptideencoded by the cDNA contained in ATCC Deposit No. 97689 on Aug. 22,1996, or by the cDNA contained in the plasmid deposited as ATCC DepositNo. 97483 on Mar. 15, 1996. TL5 may be produced through recombinantexpression of nucleic acids encoding the polypeptides of SEQ ID NO:2 orSED ID NO:4 (e.g., the cDNAs in the ATCC Deposit Numbers 97689 or 97483,respectively). Antibodies of the invention may specifically bind TL5 aswell as fragments and variants thereof, and are described in more detailbelow.

TL5 Polypeptides

In certain embodiments of the present invention, the antibodies of thepresent invention bind TL5 polypeptide, or fragments or variantsthereof. The following section describes the TL5 polypeptides, fragmentsand variants that antibodies of the invention may bind in more detail.TL5 polypeptides which the antibodies of the invention may bind aredescribed in more detail in International Publication Numbers WO97/34911and WO00/53223, each of which are herein incorporated by reference intheir entireties.

In certain embodiments, the antibodies of the present inventionspecifically bind TL5 polypeptide. An antibody that specifically bindsTL5 may, in some embodiments, bind fragments, variants (includingspecies orthologs and allelic variants of TL5), multimers or modifiedforms of TL5. For example, an antibody specific for TL5 may bind the TL5moiety of a fusion protein comprising all or a portion of TL5.

TL5 proteins may be found as monomers or multimers (i.e., dimers,trimers, tetramers, and higher multimers). Accordingly, the presentinvention relates to antibodies that bind TL5 proteins found as monomersor as part of multimers. In specific embodiments, antibodies of theinvention bind TL5 monomers, dimers, trimers or tetramers. In additionalembodiments, antibodies of the invention bind at least dimers, at leasttrimers, or at least tetramers containing one or more TL5 polypeptides.

Antibodies of the invention may bind TL5 homomers or heteromers. As usedherein, the term homomer, refers to a multimer containing only TL5proteins of the invention (including TL5 fragments, variants, and fusionproteins, as described herein). These homomers may contain TL5 proteinshaving identical or different polypeptide sequences. In a specificembodiment, a homomer of the invention is a multimer containing only TL5proteins having an identical polypeptide sequence. In another specificembodiment, antibodies of the invention bind TL5 homomers containing TL5proteins having different polypeptide sequences. In specificembodiments, antibodies of the invention bind a TL5 homodimer (e.g.,containing TL5 proteins having identical or different polypeptidesequences) or a homotrimer (e.g., containing TL5 proteins havingidentical or different polypeptide sequences). In additionalembodiments, antibodies of the invention bind at least a homodimer, atleast a homotrimer, or at least a homotetramer of TL5.

As used herein, the term heteromer refers to a multimer containingheterologous proteins (i.e., proteins containing polypeptide sequencesthat do not correspond to a polypeptide sequences encoded by a geneencoding TL5) in addition to the TL5 proteins of the invention. In aspecific embodiment, antibodies of the invention bind a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, theantibodies of the invention bind at least a heterodimer, at least aheterotrimer, or at least a heterotetramer containing one or more TL5polypeptides.

Antibodies of the invention may bind TL5 polypeptide multimers that arethe result of hydrophobic, hydrophilic, ionic and/or covalentassociations and/or may be indirectly linked, by for example, liposomeformation. Thus, in one embodiment, antibodies of the invention may bindmultimers, such as, for example, homodimers or homotrimers, that areformed when TL5 proteins contact one another in solution. In anotherembodiment, antibodies of the invention may bind heteromultimers, suchas, for example, heterotrimers or heterotetramers, that are formed whenproteins of the invention contact antibodies to the TL5 polypeptides(including antibodies to the heterologous polypeptide sequence in afusion protein) in solution. In other embodiments, multimers that one ormore antibodies of the invention may bind, are formed by covalentassociations with and/or between the TL5 proteins of the invention. Suchcovalent associations may involve one or more amino acid residuescontained in the polypeptide sequence of the protein (e.g., thepolypeptide sequence recited in SEQ ID NO:2 or the polypeptide encodedby the deposited cDNA clone of ATCC Deposit 97689). In one instance, thecovalent associations are cross-linking between cysteine residueslocated within the polypeptide sequences of the proteins which interactin the native (i.e., naturally occurring) polypeptide. In anotherinstance, the covalent associations are the consequence of chemical orrecombinant manipulation. Alternatively, such covalent associations mayinvolve one or more amino acid residues contained in the heterologouspolypeptide sequence in a TL5 fusion protein. In one example, covalentassociations are between the heterologous sequence contained in a fusionprotein (see, e.g., U.S. Pat. No. 5,478,925). In a specific example, thecovalent associations are between the heterologous sequence contained ina TL5-Fc fusion protein (as described herein). In another specificexample, covalent associations of fusion proteins are betweenheterologous polypeptide sequences from another TNF familyligand/receptor member that is capable of forming covalently associatedmultimers, such as for example, oseteoprotegerin (see, e.g.,International Publication No. WO 98/49305, the contents of which areherein incorporated by reference in its entirety).

Antibodies of the invention may bind TL5 polypeptide multimers that weregenerated using chemical techniques known in the art. For example,proteins desired to be contained in the multimers of the invention maybe chemically cross-linked using linker molecules and linker moleculelength optimization techniques known in the art (see, e.g., U.S. Pat.No. 5,478,925, which is herein incorporated by reference in itsentirety). Additionally, multimers that may be bound by one or moreantibodies of the invention may be generated using techniques known inthe art to form one or more inter-molecule cross-links between thecysteine residues located within the polypeptide sequence of theproteins desired to be contained in the multimer (see, e.g., U.S. Pat.No. 5,478,925, which is herein incorporated by reference in itsentirety). Further, proteins that may be bound by one or more antibodiesof the invention may be routinely modified by the addition of cysteineor biotin to the C terminus or N-terminus of the polypeptide sequence ofthe protein and techniques known in the art may be applied to generatemultimers containing one or more of these modified proteins (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Additionally, techniques known in the art may be appliedto generate liposomes containing the protein components desired to becontained in the multimer that one or more antibodies of the inventionmay bind (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

Alternatively, antibodies of the invention may bind TL5 proteinmultimers that were generated using genetic engineering techniques knownin the art. In one embodiment, proteins contained in multimers that maybe bound by one or more antibodies of the invention are producedrecombinantly using fusion protein technology described herein orotherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety). In a specificembodiment, polynucleotides coding for a homodimer that may be bound byone or more antibodies of the invention are generated by ligating apolynucleotide sequence encoding a TL5 polypeptide to a sequenceencoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus (see,e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by referencein its entirety). In another embodiment, recombinant techniquesdescribed herein or otherwise known in the art are applied to generaterecombinant TL5 polypeptides which contain a transmembrane domain andwhich can be incorporated by membrane reconstitution techniques intoliposomes (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). In another embodiment, twoor more TL5 polypeptides are joined through synthetic linkers (e.g.,peptide, carbohydrate or soluble polymer linkers). Examples includethose peptide linkers described in U.S. Pat. No. 5,073,627 (herebyincorporated by reference). Proteins comprising multiple TL5polypeptides separated by peptide linkers may be produced usingconventional recombinant DNA technology. In specific embodiments,antibodies of the invention bind proteins comprising multiple TL5polypeptides separated by peptide linkers.

Another method for preparing multimer TL5 polypeptides involves use ofTL5 polypeptides fused to a leucine zipper or isoleucine polypeptidesequence. Leucine zipper domains and isoleucine zipper domains arepolypeptides that promote multimerization of the proteins in which theyare found. Leucine zippers were originally identified in severalDNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), andhave since been found in a variety of different proteins. Among theknown leucine zippers are naturally occurring peptides and derivativesthereof that dimerize or trimerize. Examples of leucine zipper domainssuitable for producing soluble multimeric TL5 proteins are thosedescribed in PCT application WO 94/10308, hereby incorporated byreference. Recombinant fusion proteins comprising a soluble TL5polypeptide fused to a peptide that dimerizes or trimerizes in solutionare expressed in suitable host cells, and the resulting solublemultimeric TL5 is recovered from the culture supernatant usingtechniques known in the art. In specific embodiments, antibodies of theinvention bind TL5-leucine zipper fusion protein monomers and/orTL5-leucine zipper fusion protein multimers.

Certain members of the TNF family of proteins are believed to exist intrimeric form (Beutler and Huffel, Science 264:667, 1994; Banner et al.,Cell 73:431, 1993). Thus, trimeric TL5 may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties arethose that preferentially form trimers. One example is a leucine zipperderived from lung surfactant protein D (SPD), as described in Hoppe etal. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser.No. 08/446,922, hereby incorporated by reference. In specificembodiments, antibodies of the invention bind TL5-leucine zipper fusionprotein trimers.

Other peptides derived from naturally occurring trimeric proteins may beemployed in preparing trimeric TL5. In specific embodiments, antibodiesof the invention bind TL5-fusion protein monomers and/or TL5 fusionprotein trimers.

Antibodies that bind TL5 may bind them as isolated polypeptides or intheir naturally occurring state. For, example antibodies of the presentinvention may bind recombinantly produced TL5. In a specific embodiment,antibodies of the present invention bind TL5 expressed on the surface ofa cell (e.g., a T cell or a T cell line) wherein the TL5 protein inencoded by a polynucleotide encoding amino acids 1 to 240 of SEQ ID NO:2operably associated with a regulatory sequence that controls expressionof said polynucleotide.

Antibodies of the present invention may bind TL5 polypeptide fragmentscomprising or alternatively, consisting of, an amino acid sequencecontained in SEQ ID NO:2, encoded by the cDNA contained in ATCC depositNumber 97689, or encoded by nucleic acids which hybridize (e.g., understringent hybridization conditions) to the nucleotide sequence containedin ATCC deposit Number 97689, or the complementary strand thereto.Protein fragments may be “free-standing,” or comprised within a largerpolypeptide of which the fragment forms a part or region, mostpreferably as a single continuous region. Antibodies of the presentinvention may bind polypeptide fragments, including, for example,fragments that comprise or alternatively, consist of from about aminoacid residues: 1 to 23, 24 to 43, 44 to 63, 64 to 83, 84 to 103, 104 to123, 124 to 143, 144 to 163, 164 to 183, 184 to 203, 204 to 223, and/or224 to 240 of SEQ ID NO:2. In this context “about” includes theparticularly recited value, larger or smaller by several (5, 4, 3, 2,or 1) amino acids, at either extreme or at both extremes. Moreover,polypeptide fragments that antibodies of the invention may bind can beat least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 175 or 200 amino acids in length. In this context “about”includes the particularly recited value, larger or smaller by several(5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.

Preferably, antibodies of the present invention bind polypeptidefragments selected from the group: a polypeptide comprising oralternatively, consisting of, the TL5 extracellular domain (predicted toconstitute amino acid residues from about 59 to about 240 in SEQ IDNO:2); a polypeptide comprising or alternatively, consisting of, the TL5transmembrane domain (predicted to constitute amino acid residues fromabout 38 to about 58 in SEQ ID NO:2); a polypeptide comprising oralternatively, consisting of, fragment of the predicted mature TL5polypeptide, wherein the fragment has a TL5 functional activity (e.g.,antigenic activity or biological acitivity); a polypeptide comprising oralternatively, consisting of, the TL5 intracellular domain (predicted toconstitute amino acid residues from about 1 to about 37 in SEQ ID NO:2);and a polypeptide comprising, or alternatively, consisting of, one, two,three, four or more, epitope bearing portions of the TL5 protein. Inadditional embodiments, the polypeptide fragments of the inventioncomprise, or alternatively, consist of, any combination of 1, 2, 3, 4,or all 5 of the above members. The amino acid residues constituting theTL5 extracellular, transmembrane and intracellular domains have beenpredicted by computer analysis. Thus, as one of ordinary skill wouldappreciate, the amino acid residues constituting these domains may varyslightly (e.g., by about 1 to about 15 amino acid residues) depending onthe criteria used to define each domain. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

In another preferred embodiment, antibodies of the present inventionbind TL5 polypeptides comprising, or alternatively consisting theextracellular soluble domain of TL5 (amino acid residues 59-240 of SEQID NO:2) In highly preferred embodiments, the antibodies of theinvention that bind all or a portion of the extracellular soluble domainof TL5 prevent a TL5 receptor (e.g., TR2, TR6, LTβR) from binding toTL5. In other highly preferred embodiments, the antibodies of theinvention that bind all or a portion of the extracellular soluble domainof TL5 antagonize or neutralize TL5. In other highly preferredembodiments, the antibodies of the invention that bind all or a portionof the extracellular soluble domain of TL5 inhibit proliferation of thecells expressing a TL5 receptor. In other highly preferred embodiments,the antibodies of the invention that bind all or a portion of theextracellular soluble domain of TL5 inhibit differentiation of the cellsexpressing a TL5 receptor (e.g., T cells). In other highly preferredembodiments, the antibodies of the invention that bind all or a portionof the extracellular soluble domain of TL5 inhibit apoptosis of cellsexpressing a TL5 receptor.

Antibodies of the invention may also bind fragments comprising, oralternatively, consisting of structural or functional attributes of TL5.Such fragments include amino acid residues that comprise alpha-helix andalpha-helix forming regions (“alpha-regions”), beta-sheet andbeta-sheet-forming regions (“beta-regions”), turn and turn-formingregions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, surface forming regions,and high antigenic index regions (i.e., containing four or morecontiguous amino acids having an antigenic index of greater than orequal to 1.5, as identified using the default parameters of theJameson-Wolf program) of complete (i.e., full-length) TL5. Certainpreferred regions are those set out in Table 2 and include, but are notlimited to, regions of the aforementioned types identified by analysisof the amino acid sequence depicted in (SEQ ID NO:2), such preferredregions include; Garnier-Robson predicted alpha-regions, beta-regions,turn-regions, and coil-regions; Chou-Fasman predicted alpha-regions,beta-regions, and turn-regions; Kyte-Doolittle predicted hydrophilicregions; Eisenberg alpha and beta amphipathic regions; Eminisurface-forming regions; and Jameson-Wolf high antigenic index regions,as predicted using the default parameters of these computer programs.

The data representing the structural or functional attributes of TL5 setforth in Table 2, as described above, was generated using the variousmodules and algorithms of the DNA*STAR set on default parameters. ColumnI represents the results of a Garnier-Robson analysis of alpha helicalregions; Column II represents the results of a Chou-Fasman analysis ofalpha helical regions; Column III represents the results of a GamierRobson analysis of beta sheet regions; Column IV represents the resultsof a Chou-Fasman analysis of beta sheet regions; Column V represents theresults of a Gamier Robson analysis of turn regions; Column VIrepresents the results of a Chou-Fasman analysis of turn regions; ColumnVII represents the results of a Gamier Robson analysis of coil regions;Column VIII represents a Kyte-Doolittle hydrophilicity plot; Column;Column IX represents a Hopp-Woods hydrophobicity plot; Column Xrepresents the results of an Eisenberg analysis of alpha amphipathicregions; Column XI represents the results of an Eisenberg analysis ofbeta amphipathic regions; Column XII represents the results of aKarplus-Schultz analysis of flexible regions; Column XIII represents theJameson-Wolf antigenic index score; and Column XIV represents the Eminisurface probability plot.

In a preferred embodiment, the data presented in columns VIII, XIII, andXIV of Table 2 can be used to determine regions of TL5 which exhibit ahigh degree of potential for antigenicity. Regions of high antigenicityare determined from the data presented in columns VIII, XIII, and/or XIVby choosing values which represent regions of the polypeptide which arelikely to be exposed on the surface of the polypeptide in an environmentin which antigen recognition may occur in the process of initiation ofan immune response.

The above-mentioned preferred regions set out in Table 2 include, butare not limited to, regions of the aforementioned types identified byanalysis of the amnino acid sequence set out in SEQ ID NO:2. As set outin Table 2, such preferred regions include Garnier-Robson alpha-regions,beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions,beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions,Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexibleregions, Jameson-Wolf regions of high antigenic index and Eminisurface-forming regions. Among preferred polypeptide fragments that oneor more antibodies of the invention may bind are those that compriseregions of TL5 that combine several structural features, such as several(e.g., 1, 2, 3, or 4) of the same or different region features set outabove and in Table 2.

TABLE 2 Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV Met1 A . . . . . . 0.19 −0.71 . . . 0.95 1.49 Glu 2 A . . . . . . −0.28−0.50 * . . 0.50 0.87 Glu 3 A . . B . . . 0.22 −0.29 * . . 0.30 0.50 Ser4 A . . B . . . 0.40 −0.71 * . . 0.60 0.99 Val 5 A . . B . . . 0.49−0.90 * * . 0.60 0.89 Val 6 A . . B . . . 0.23 −0.51 . . . 0.60 0.69 Arg7 . . B . . T . −0.47 0.13 * * . 0.10 0.38 Pro 8 . . B . . T . −1.320.53 * * . −0.20 0.44 Ser 9 . . B . . T . −1.88 0.53 * . . −0.20 0.44Val 10 . . B . . T . −1.02 0.53 * * . −0.20 0.17 Phe 11 . . B . . . .−0.51 0.53 . * . −0.40 0.18 Val 12 . . B . . . . −0.62 0.53 . * . −0.120.13 Val 13 . . B . . T . −0.72 0.54 . * . 0.36 0.31 Asp 14 . . B . . T. −0.42 0.39 . * F 1.09 0.52 Gly 15 . . . . T T . −0.46 −0.40 . * F 2.521.17 Gln 16 . . . . T T . 0.03 −0.36 . * F 2.80 1.11 Thr 17 . . . B . .C 0.19 −0.57 . * F 2.22 1.03 Asp 18 . . B B . . . 0.73 0.21 . * F 0.690.90 Ile 19 . . B B . . . 0.84 0.27 . * F 0.41 0.75 Pro 20 . . B B . . .0.38 −0.13 * * . 0.73 1.02 Phe 21 . . B B . . . 0.03 0.07 * . . −0.300.50 Thr 22 . . B B . . . 0.46 0.50 * . . −0.26 0.71 Arg 23 . . B B . .. 0.16 −0.19 * . F 1.13 0.90 Leu 24 . . . B T . . 1.01 −0.23 * . F 2.021.39 Gly 25 . . . B T . . 1.33 −0.51 * . F 2.66 1.31 Arg 26 . . . . T T. 2.14 −1.00 * . F 3.40 1.31 Ser 27 . . . . T T . 2.46 −1.00 * . F 3.063.11 His 28 . . . . T T . 2.04 −1.29 * . F 3.03 5.44 Arg 29 . . . . T T. 2.19 −1.33 * . F 3.00 3.72 Arg 30 . . . . T . . 2.23 −0.76 * * F 2.771.49 Gln 31 . . . . T T . 1.27 −0.76 * . F 2.94 1.47 Ser 32 . . . . T T. 0.98 −0.61 * . F 3.10 0.56 Cys 33 . . B . . T . 1.12 −0.11 * . . 1.940.29 Ser 34 . . B . . T . 0.16 −0.11 * . . 1.63 0.32 Val 35 . . B B . .. −0.30 0.13 * * . 0.32 0.18 Ala 36 . . B B . . . −1.11 0.17 . * . 0.010.33 Arg 37 . . B B . . . −1.16 0.29 * * . −0.30 0.20 Val 38 . . B B . .. −1.30 0.33 * * . −0.30 0.27 Gly 39 . . B B . . . −1.81 0.37 * * .−0.30 0.22 Leu 40 . A B . . . . −1.77 0.56 * * . −0.60 0.09 Gly 41 . A B. . . . −1.99 1.24 * * . −0.60 0.10 Leu 42 . A B . . . . −2.91 1.29 * *. −0.60 0.09 Leu 43 . A B . . . . −2.66 1.54 . . . −0.60 0.09 Leu 44 . AB . . . . −2.66 1.47 . . . −0.60 0.09 Leu 45 . A B . . . . −2.43 1.47 .. . −0.60 0.10 Leu 46 . A B . . . . −2.43 1.29 . . . −0.60 0.13 Met 47 AA . . . . . −2.43 1.03 . . . −0.60 0.15 Gly 48 A . . . . T . −2.21 1.03. . . −0.20 0.15 Ala 49 A . . . . T . −2.26 0.84 . . . −0.20 0.19 Gly 50A . . . . T . −1.44 0.80 . . . −0.20 0.14 Leu 51 A . . . . T . −0.980.59 . . . −0.20 0.25 Ala 52 . A B . . . . −0.67 0.59 . * . −0.60 0.24Val 53 A A . . . . . −1.02 1.00 . . . −0.60 0.26 Gln 54 . A B . . . .−1.24 1.36 . * . −0.60 0.27 Gly 55 . A B . . . . −1.71 1.36 . * . −0.600.22 Trp 56 A A . . . . . −0.90 1.54 . * . −0.60 0.24 Phe 57 . A B . . .. −1.12 1.30 . * . −0.60 0.24 Leu 58 . A B . . . . −0.30 1.59 . * .−0.60 0.20 Leu 59 . A B . . . . −0.59 1.66 * * . −0.60 0.26 Gln 60 . A B. . . . −0.13 1.66 * * . −0.60 0.32 Leu 61 . A . . . . C −0.66 0.87 * *. −0.40 0.76 His 62 . A . . . . C −0.30 0.87 * * . −0.40 0.76 Trp 63 . A. . . . C 0.51 0.61 * * . −0.40 0.43 Arg 64 A A . . . . . 0.72 0.21 * *. −0.30 0.91 Leu 65 A A . . . . . −0.13 0.14 * * . −0.30 0.66 Gly 66 . A. . T . . 0.37 0.29 * * . 0.10 0.47 Glu 67 . A B . . . . 0.51 −0.14 * *. 0.30 0.34 Met 68 . A B . . . . −0.01 −0.14 * * . 0.30 0.82 Val 69 . AB . . . . −0.33 −0.14 * . . 0.64 0.68 Thr 70 . A B . . . . 0.48 −0.14 *. . 0.98 0.61 Arg 71 . A B . . . . 0.48 −0.14 * * F 1.62 1.02 Leu 72 . .B . . T . 0.27 −0.33 * . F 2.36 1.37 Pro 73 . . . . T T . 0.28 −0.54 * .F 3.40 1.46 Asp 74 . . . . T T . 0.79 −0.53 * . F 2.91 0.75 Gly 75 . . .. . T C 0.80 −0.10 * . F 2.07 0.91 Pro 76 . . . . . T C 0.40 −0.40 * . F1.73 0.78 Ala 77 . . . . . T C 1.21 0.09 . . F 0.79 0.49 Gly 78 . . . .. T C 1.42 0.09 . . F 0.45 0.86 Ser 79 . . . . . T C 0.61 0.06 * . F0.45 0.97 Trp 80 A A . . . . . 0.07 0.31 * . F −0.15 0.79 Glu 81 A A . .. . . 0.28 0.50 * . . −0.60 0.56 Gln 82 A A . . . . . 0.87 0.47 . * .−0.60 0.72 Leu 83 A A . . . . . 1.32 0.09 . . . −0.15 1.19 Ile 84 A A .. . . . 1.73 −0.83 . . . 0.75 1.35 Gln 85 A A . . . . . 1.72 −0.83 . . F0.90 1.52 Glu 86 A A . . . . . 1.69 −0.84 . . F 0.90 2.48 Arg 87 A A . .. . . 1.69 −1.03 . . F 0.90 4.81 Arg 88 . A . . T . . 1.64 −1.71 . . F1.30 4.81 Ser 89 . A . . T . . 2.53 −1.47 . . F 1.30 2.06 His 90 . A . .. . C 2.32 −1.07 . . . 0.95 1.69 Glu 91 . A . . . . C 1.73 −0.64 * . .0.95 1.34 Val 92 . A . . . . C 1.03 −0.14 * . . 0.65 1.01 Asn 93 . . . .. T C 0.89 −0.03 . * . 0.90 0.75 Pro 94 A . . . . T . 0.38 −0.03 . * .0.70 0.59 Ala 95 A . . . . T . 0.10 0.66 . * . −0.20 0.65 Ala 96 A . . .. T . −0.24 0.50 . * . −0.20 0.59 His 97 A . . . . . . 0.02 0.53 . * .−0.40 0.37 Leu 98 A . . . . . . 0.02 0.60 . . . −0.40 0.37 Thr 99 . . B. . . . −0.07 0.50 . . . −0.40 0.60 Gly 100 . . . . . . C 0.22 0.39 . .F 0.25 0.59 Ala 101 . . . . . . C 0.00 0.27 . . F 0.25 0.96 Asn 102 . .B . . T . −0.28 0.27 . . F 0.25 0.55 Ser 103 . . B . . T . 0.19 0.27 . .F 0.25 0.80 Ser 104 . . B . . T . 0.20 0.27 . * F 0.25 0.78 Leu 105 . .B . . T . 0.20 0.16 . . F 0.25 0.65 Thr 106 . . B . . . . 0.44 0.19 . *F 0.05 0.48 Gly 107 . . . . T T . 0.23 0.23 . . F 0.65 0.35 Ser 108 . .. . T T . −0.28 0.27 . . F 0.65 0.66 Gly 109 . . . . . T C −0.79 0.27 .. F 0.45 0.38 Gly 110 . . . . . T C −0.27 0.47 . . F 0.15 0.32 Pro 111 .A . . . . C 0.04 0.96 . . F −0.25 0.25 Leu 112 . A . . . . C 0.08 0.57 .. F −0.25 0.43 Leu 113 . A B . . . . 0.38 0.63 . * . −0.60 0.63 Trp 114. A B . . . . −0.09 0.60 . . . −0.60 0.71 Glu 115 . A B . . . . −0.090.86 . * . −0.60 0.71 Thr 116 A A . . . . . −0.69 0.60 . * F −0.45 0.85Gln 117 A A . . . . . −0.47 0.60 . * F −0.45 0.67 Leu 118 A A . . . . .−0.36 0.19 . . . −0.30 0.39 Gly 119 A A . . . . . −0.88 0.97 * * . −0.600.23 Leu 120 A A . . . . . −0.77 1.17 * * . −0.60 0.11 Ala 121 . A B . .. . −0.80 0.77 * . . −0.60 0.26 Phe 122 . A B . . . . −1.61 0.51 * . .−0.60 0.26 Leu 123 . A B . . . . −1.10 0.77 * . . −0.60 0.26 Arg 124 . AB . . . . −1.00 0.47 * . . −0.60 0.35 Gly 125 . A B . . . . −0.22 0.73 .. . −0.60 0.63 Leu 126 . . B . . . . 0.37 0.44 * . . −0.25 1.05 Ser 127. . . . . . C 0.72 −0.24 * . . 0.70 0.89 Tyr 128 . . . . . . C 0.940.19 * * . 0.10 0.89 His 129 . . . . T T . 0.02 0.26 * . . 0.65 1.09 Asp130 . . . . T T . −0.49 0.26 . . . 0.50 0.67 Gly 131 . . B . . T . −0.530.51 . . . −0.20 0.32 Ala 132 . . B . . T . −0.54 0.40 * . . −0.20 0.17Leu 133 . . B B . . . −0.26 0.39 * . . −0.30 0.15 Val 134 . . B B . . .−0.81 0.39 * . . −0.30 0.30 Val 135 . . B B . . . −1.16 0.46 * . . −0.600.30 Thr 136 . . B B . . . −1.06 0.39 . . . −0.30 0.36 Lys 137 . . B . .T . −0.71 0.46 . . F −0.05 0.77 Ala 138 . . B . . T . −0.14 0.57 . . .−0.05 1.62 Gly 139 . . B . . T . −0.18 0.69 . . . −0.05 1.76 Tyr 140 . .B . . T . 0.43 0.89 * . . −0.20 0.62 Tyr 141 . . B B . . . 0.44 1.64 . .. −0.60 0.96 Tyr 142 . . B B . . . 0.44 1.53 . * . −0.45 1.30 Ile 143 .. B B . . . 0.18 1.10 . * . −0.45 1.66 Tyr 144 . . B B . . . 0.52 0.99. * . −0.60 0.78 Ser 145 . . B B . . . −0.04 0.63 . * . −0.60 0.87 Lys146 . . B B . . . −0.14 0.56 . * . −0.45 1.02 Val 147 . . B B . . .−0.24 0.30 . * . −0.30 0.64 Gln 148 . . B B . . . −0.21 −0.03 . * . 0.300.48 Leu 149 . . B B . . . −0.31 0.23 . * . −0.30 0.18 Gly 150 . . B B .. . −0.68 0.66 . * . −0.60 0.24 Gly 151 . . B B . . . −0.93 0.59 . * .−0.60 0.07 Val 152 . . B B . . . −0.89 0.61 . . . −0.60 0.14 Gly 153 . .B . . . . −1.23 0.61 . . . −0.40 0.11 Cys 154 . . B . . T . −1.23 0.61 .. . −0.20 0.11 Pro 155 . . B . . T . −1.48 0.87 . . . −0.20 0.13 Leu 156. . B . . T . −1.43 0.73 . . . −0.20 0.13 Gly 157 . . B . . T . −0.890.69 . . . −0.20 0.32 Leu 158 . . B B . . . −1.43 0.60 . . . −0.60 0.30Ala 159 . . B B . . . −1.08 0.86 . . . −0.60 0.26 Ser 160 . . B B . . .−0.90 0.66 . . . −0.60 0.37 Thr 161 . . B B . . . −0.43 0.73 * . F −0.450.62 Ile 162 . . B B . . . −0.90 0.47 * . . −0.60 0.60 Thr 163 . . B B .. . −0.33 0.66 * . . −0.60 0.37 His 164 . . B B . . . 0.30 1.03 * . .−0.60 0.40 Gly 165 . . B B . . . 0.71 0.54 . . . −0.45 1.15 Leu 166 . .B B . . . 0.71 −0.14 . . . 0.75 1.56 Tyr 167 . . . B T . . 1.39 −0.14 *. . 1.45 1.66 Lys 168 . . . B T . . 1.81 −0.21 * . F 1.90 2.59 Arg 169 .. B . . . . 1.60 −0.64 * . F 2.30 6.15 Thr 170 . . . . . T C 1.73−0.57 * . F 3.00 6.15 Pro 171 . . . . . T C 2.54 −0.90 * . F 2.70 4.75Arg 172 . . . . . T C 2.79 −0.90 * . F 2.40 4.20 Tyr 173 . . . . . T C1.93 −0.90 * * F 2.10 5.05 Pro 174 . A . . . . C 1.82 −0.70 * * F 1.402.69 Glu 175 A A . . . . . 1.32 −1.13 * * F 0.90 2.38 Glu 176 A A . . .. . 0.72 −0.44 * * F 0.60 1.25 Leu 177 A A . . . . . −0.24 −0.51 * * .0.60 0.67 Glu 178 A A . . . . . −0.30 −0.30 * . . 0.30 0.29 Leu 179 A A. . . . . −0.09 0.09 . . . −0.30 0.22 Leu 180 A A . . . . . −0.09 0.49 .. . −0.60 0.47 Val 181 A A . . . . . −0.39 0.20 . * . −0.30 0.47 Ser 182A A . . . . . 0.21 0.59 . . F −0.45 0.76 Gln 183 . . . . T . . −0.460.33 . . F 0.60 1.42 Gln 184 . . B . . . . 0.01 0.21 . * F 0.20 1.02 Ser185 . . . . . T C 0.93 0.00 * * F 0.45 0.76 Pro 186 . . . . T T . 1.20−0.39 . * F 1.25 0.85 Cys 187 . . . . T T . 1.19 −0.29 . * F 1.25 0.50Gly 188 . . B . . T . 0.89 −0.20 . * F 1.15 0.54 Arg 189 . . B B . . .0.59 −0.20 . * F 1.05 0.47 Ala 190 . . B B . . . 0.59 −0.24 * * F 1.501.16 Thr 191 . . . B . . C 0.91 −0.43 . * F 2.00 1.58 Ser 192 . . . . .T C 0.72 −0.86 . * F 3.00 1.58 Ser 193 . . B . . T . 0.78 −0.21 . * F2.20 1.16 Ser 194 . . B . . T . 0.38 0.20 * * F 1.15 0.84 Arg 195 . . B. . T . 0.97 0.63 * * F 0.55 0.66 Val 196 . . B B . . . 0.98 0.24 * * .0.00 0.82 Trp 197 . . . B T . . 0.98 0.24 . * . 0.10 0.82 Trp 198 . . BB . . . 0.58 0.24 . * . −0.30 0.56 Asp 199 . . B . . T . 0.07 1.03 . * F−0.05 0.66 Ser 200 . . . . . T C −0.39 1.07 . * F 0.15 0.52 Ser 201 . .. . . T C 0.12 0.59 . . F 0.15 0.49 Phe 202 . . . . T T . −0.44 0.10 . .F 0.65 0.29 Leu 203 . . . B . . C −1.01 0.74 . . . −0.40 0.16 Gly 204 .. . B . . C −1.04 1.00 . . . −0.40 0.09 Gly 205 . . . B . . C −1.56 1.11. . . −0.40 0.14 Val 206 . A B . . . . −1.26 1.01 . * . −0.60 0.14 Val207 . A B . . . . −1.14 0.33 . . . −0.30 0.24 His 208 A A . . . . .−0.68 0.40 . . . −0.60 0.25 Leu 209 A A . . . . . −0.33 0.40 . . . −0.600.33 Glu 210 A A . . . . . 0.01 −0.24 . * . 0.30 0.77 Ala 211 A A . . .. . 0.01 −0.89 . . F 0.75 0.98 Gly 212 A A . . . . . 0.01 −0.74 . * F0.75 0.88 Glu 213 A A . B . . . −0.81 −0.79 * * F 0.75 0.38 Glu 214 A A. B . . . 0.11 −0.14 * * F 0.45 0.28 Val 215 A A . B . . . −0.74−0.64 * * . 0.60 0.55 Val 216 A A . B . . . −0.97 −0.43 * * . 0.30 0.24Val 217 A A . B . . . −0.62 0.26 * * . −0.30 0.11 Arg 218 A A . B . . .−0.62 0.26 * * . −0.30 0.25 Val 219 A A . B . . . −0.51 −0.39 * * . 0.300.59 Leu 220 A A . B . . . −0.47 −1.03 * . . 0.75 1.56 Asp 221 A A . B .. . −0.47 −0.99 * . F 0.75 0.66 Glu 222 A A . . . . . 0.50 −0.34 * . F0.45 0.66 Arg 223 A A . . . . . −0.42 −0.99 * * F 0.90 1.56 Leu 224 A A. . . . . 0.54 −0.99 * * . 0.60 0.77 Val 225 . A B . . . . 1.36−0.99 * * . 0.94 0.87 Arg 226 . A B . . . . 1.01 −0.99 . * . 1.28 0.74Leu 227 . . B . . T . 0.70 −0.56 * * . 2.02 0.89 Arg 228 . . B . . T .0.70 −0.76 . * F 2.66 1.73 Asp 229 . . . . T T . 1.21 −1.40 * * F 3.401.73 Gly 230 . . . . T T . 1.82 −1.01 * * F 3.06 2.81 Thr 231 . . . . T. . 1.01 −0.94 * * F 2.52 2.25 Arg 232 . . B . . . . 1.48 −0.16 * * F1.48 1.17 Ser 233 . . B . . T . 0.78 0.27 * * F 0.74 1.17 Tyr 234 . . B. . T . 0.08 0.34 * . . 0.10 0.82 Phe 235 . . B . . T . −0.18 0.64 . . .−0.20 0.36 Gly 236 . . B . . T . −0.72 1.26 . . . −0.20 0.27 Ala 237 . AB . . . . −1.22 1.51 . . . −0.60 0.13 Phe 238 . A B . . . . −1.31 1.19 .. . −0.60 0.19 Met 239 . A B . . . . −1.46 0.83 . . . −0.60 0.24 Val 240. A B . . . . −1.14 0.83 . . . −0.60 0.30

In another aspect, the invention provides an antibody that binds apeptide or polypeptide comprising an epitope-bearing portion of apolypeptide described herein. The epitope of this polypeptide portion isan immunogenic or antigenic epitope of a polypeptide of the invention.An “immunogenic epitope” is defined as a part of a protein that elicitsan antibody response when the whole protein is the immunogen. On theother hand, a region of a protein molecule to which an antibody can bindis defined as an “antigenic epitope.” The number of immunogenic epitopesof a protein generally is less than the number of antigenic epitopes.See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA81:3998-4002 (1983).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M.,Green, N. and Learner, R. A. (1983) Antibodies that react withpredetermined sites on proteins. Science 219:660-666. Peptides capableof eliciting protein-reactive sera are frequently represented in theprimary sequence of a protein, can be characterized by a set of simplechemical rules, and are confined neither to immunodominant regions ofintact proteins nor to the amino or carboxyl terminals.

Antigenic epitope-bearing peptides and polypeptides are therefore usefulto raise antibodies, including monoclonal antibodies, that bind to a TL5polypeptide of the invention. See, for instance, Wilson et al., Cell37:767-778 (1984) at 777. Antigenic epitope-bearing peptides andpolypeptides preferably contain a sequence of at least seven, morepreferably at least nine and most preferably between at least about 15to about 30 amino acids contained within the amino acid sequence of SEQID NO:2.

Antibodies of the invention may bind one or more antigenic TL5polypeptides or peptides including, but not limited to: a polypeptidecomprising amino acid residues from about 24 to about 34 of SEQ ID NO:2;a polypeptide comprising amino acid residues from about 71 to about 76of SEQ ID NO:2; a polypeptide comprising amino acid residues from about168 to about 173 of SEQ ID NO:2; a polypeptide comprising amino acidresidues from about 190 to about 193 of SEQ ID NO:2; and/or apolypeptide comprising amino acid residues from about 227 to about 231of SEQ ID NO:2. In this context “about” includes the particularlyrecited range, larger or smaller by several (5, 4, 3, 2, or 1) aminoacids, at either terminus or at both termini. As indicated above, theinventors have determined that the above polypeptide fragments areantigenic regions of the TL5 protein. Epitope-bearing TL5 peptides andpolypeptides may be produced by any conventional means. Houghten, R. A.,“General method for the rapid solid-phase synthesis of large numbers ofpeptides: specificity of antigen-antibody interaction at the level ofindividual amino acids,” Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985).This “Simultaneous Multiple Peptide Synthesis (SMPS)” process is furtherdescribed in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).

As one of skill in the art will appreciate, TL5 polypeptides and theepitope-bearing fragments thereof described herein (e.g., correspondingto all or a portion of the extracellular domain such as, for example,amino acid residues 59 to 240 of SEQ ID NO:2) can be combined with partsof the constant domain of immunoglobulins (IgG), resulting in chimericpolypeptides. These fusion proteins facilitate purification and show anincreased half-life in vivo. This has been shown, e.g., for chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins (EPA 394,827; Traunecker etal., Nature 331:84-86 (1988)). Fusion proteins that have adisulfide-linked dimeric structure due to the IgG part can also be moreefficient in binding and neutralizing other molecules than the monomericTL5 protein or protein fragment alone (Fountoulakis et al., J Biochem270:3958-3964 (1995)). Thus, antibodies of the invention may bind fusionproteins that comprise all or a portion of a TL5 polypeptide.

Recombinant DNA technology known to those skilled in the art can be usedto create novel mutant proteins or “muteins” including single ormultiple amino acid substitutions, deletions, additions or fusionproteins. Such modified polypeptides can show, e.g., enhanced activityor increased stability. In addition, they may be purified in higheryields and show better solubility than the corresponding naturalpolypeptide, at least under certain purification and storage conditions.Antibodies of the present invention may also bind such modified TL5polypeptides or TL5 polypeptide fragments or variants.

For instance, for many proteins, including the extracellular domain of amembrane associated protein or the mature form(s) of a secreted protein,it is known in the art that one or more amino acids may be deleted fromthe N-terminus or C-terminus without substantial loss of biologicalfunction, or loss of the ability to be bound by a specific antibody. Forinstance, Ron et al., J. Biol. Chem., 268:2984-2988 (1993) reportedmodified KGF proteins that had heparin binding activity even if 3, 8, or27 amino-terminal amino acid residues were missing.

However, even if deletion of one or more amino acids from the N-terminusof a protein results in modification or loss of one or more biologicalfunctions of the protein, other functional activities (e.g., biologicalactivities, ability to multimerize, ability to bind a TL5 receptor(e.g., TR2, TR6, or LTβR)) may still be retained. For example, theability of shortened TL5 polypeptides to induce and/or bind toantibodies which recognize the complete or mature forms of the TL5polypeptides generally will be retained when less than the majority ofthe residues of the complete or mature polypeptide are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof a complete polypeptide retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. It is not unlikely that a TL5 polypeptide with a largenumber of deleted N-terminal amino acid residues may retain somebiological or immunogenic activities. In fact, peptides composed of asfew as six TL5 amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides antibodies that bindpolypeptides having one or more residues deleted from the amino terminusof the TL5 amino acid sequence of SEQ ID NO:2 up to the glycine residueat position number 235 and polynucleotides encoding such polypeptides.In particular, the present invention provides antibodies that bindpolypeptides comprising the amino acid sequence of residues n¹-240 ofSEQ ID NO:2, where n¹ is an integer from 2 to 235 corresponding to theposition of the amino acid residue in SEQ ID NO:2.

More in particular, the invention provides antibodies that bindpolypeptides comprising, or alternatively consisting of, the amino acidsequence of residues of E-2 to V-240; E-3 to V-240; S-4 to V-240; V-5 toV-240; V-6 to V-240; R-7 to V-240; P-8 to V-240; S-9 to V-240; V-10 toV-240; F-11 to V-240; V-12 to V-240; V-13 to V-240; D-14 to V-240; G-15to V-240; Q-16 to V-240; T-17 to V-240; D-18 to V-240; I-19 to V-240;P-20 to V-240; F-21 to V-240; T-22 to V-240; R-23 to V-240; L-24 toV-240; G-25 to V-240; R-26 to V-240; S-27 to V-240; H-28 to V-240; R-29to V-240; R-30 to V-240; Q-31 to V-240; S-32 to V-240; C-33 to V-240;S-34 to V-240; V-35 to V-240; A-36 to V-240; R-37 to V-240; V-38 toV-240; G-39 to V-240; L-40 to V-240; G-41 to V-240; L-42 to V-240; L-43to V-240; L-44 to V-240; L-45 to V-240; L-46 to V-240; M-47 to V-240;G-48 to V-240; A-49 to V-240; G-50 to V-240; L-51 to V-240; A-52 toV-240; V-53 to V-240; Q-54 to V-240; G-55 to V-240; W-56 to V-240; F-57to V-240; L-58 to V-240; L-59 to V-240; Q-60 to V-240; L-61 to V-240;H-62 to V-240; W-63 to V-240; R-64 to V-240; L-65 to V-240; G-66 toV-240; E-67 to V-240; M-68 to V-240; V-69 to V-240; T-70 to V-240; R-71to V-240; L-72 to V-240; P-73 to V-240; D-74 to V-240; G-75 to V-240;P-76 to V-240; A-77 to V-240; G-78 to V-240; S-79 to V-240; W-80 toV-240; E-81 to V-240; Q-82 to V-240; L-83 to V-240; 1-84 to V-240; Q-85to V-240; E-86 to V-240; R-87 to V-240; R-88 to V-240; S-89 to V-240;H-90 to V-240; E-91 to V-240; V-92 to V-240; N-93 to V-240; P-94 toV-240; A-95 to V-240; A-96 to V-240; H-97 to V-240; L-98 to V-240; T-99to V-240; G-100 to V-240; A-101 to V-240; N-102 to V-240; S-103 toV-240; S-104 to V-240; L-105 to V-240; T-106 to V-240; G-107 to V-240;S-108 to V-240; G-109 to V-240; G-110 to V-240; P-111 to V-240; L-112 toV-240; L-113 to V-240; W-114 to V-240; E-115 to V-240; T-116 to V-240;Q-117 to V-240; L-118 to V-240; G-119 to V-240; L-120 to V-240; A-121 toV-240; F-122 to V-240; L-123 to V-240; R-124 to V-240; G-125 to V-240;L-126 to V-240; S-127 to V-240; Y-128 to V-240; H-129 to V-240; D-130 toV-240; G-131 to V-240; A-132 to V-240; L-133 to V-240; V-134 to V-240;V-135 to V-240; T-136 to V-240; K-137 to V-240; A-138 to V-240; G-139 toV-240; Y-140 to V-240; Y-141 to V-240; Y-142 to V-240; 1-143 to V-240;Y-144 to V-240; S-145 to V-240; K-146 to V-240; V-147 to V-240; Q-148 toV-240; L-149 to V-240; G-150 to V-240; G-151 to V-240; V-152 to V-240;G-153 to V-240; C-154 to V-240; P-155 to V-240; L-156 to V-240; G-157 toV-240; L-158 to V-240; A-159 to V-240; S-160 to V-240; T-161 to V-240;I-162 to V-240; T-163 to V-240; H-164 to V-240; G-165 to V-240; L-166 toV-240; Y-167 to V-240; K-168 to V-240; R-169 to V-240; T-170 to V-240;P-171 to V-240; R-172 to V-240; Y-173 to V-240; P-174 to V-240; E-175 toV-240; E-176 to V-240; L-177 to V-240; E-178 to V-240; L-179 to V-240;L-180 to V-240; V-181 to V-240; S-182 to V-240; Q-183 to V-240; Q-184 toV-240; S-185 to V-240; P-186 to V-240; C-187 to V-240; G-188 to V-240;R-189 to V-240; A-190 to V-240; T-191 to V-240; S-192 to V-240; S-193 toV-240; S-194 to V-240; R-195 to V-240; V-196 to V-240; W-197 to V-240;W-198 to V-240; D-199 to V-240; S-200 to V-240; S-201 to V-240; F-202 toV-240; L-203 to V-240; G-204 to V-240; G-205 to V-240; V-206 to V-240;V-207 to V-240; H-208 to V-240; L-209 to V-240; E-210 to V-240; A-211 toV-240; G-212 to V-240; E-213 to V-240; E-214 to V-240; V-215 to V-240;V-216 to V-240; V-217 to V-240; R-218 to V-240; V-219 to V-240; L-220 toV-240; D-221 to V-240; E-222 to V-240; R-223 to V-240; L-224 to V-240;V-225 to V-240; R-226 to V-240; L-227 to V-240; R-228 to V-240; D-229 toV-240; G-230 to V-240; T-231 to V-240; R-232 to V-240; S-233 to V-240;Y-234 to V-240; and/or F-235 to V-240 of the TL5 sequence of SEQ IDNO:2.

As mentioned above, even if deletion of one or more amino acids from theC-terminus of a protein results in modification of loss of one or morebiological functions of the protein, other functional activities (e.g.,biological activities, ability to multimerize, ability to bind a TL5(e.g., TR2, TR6, or LTβR)) may still be retained. For example theability of the shortened TL5 polypeptide to induce and/or bind toantibodies which recognize the complete or mature forms of the TL5polypeptide generally will be retained when less than the majority ofthe residues of the complete or mature polypeptide are removed from theC-terminus. Whether a particular polypeptide lacking C-terminal residuesof a complete polypeptide retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. It is not unlikely that a TL5 polypeptide with a largenumber of deleted C-terminal amino acid residues may retain somebiological or immunogenic activities. In fact, peptides composed of asfew as six TL5 amino acid residues may often evoke an immune response.

In another embodiment, antibodies of the invention bind C-terminaldeletions of the TL5 polypeptide that can be described by the generalformula 1-m¹ where m¹ is a number from 6 to 239 corresponding to theamino acid sequence identified of SEQ ID NO:2. In specific embodiments,the invention provides antibodies that bind TL5 polypeptides comprising,or alternatively consisting of, the amino acid sequence of residues: M-1to M-239; M-1 to F-238; M-1 to A-237; M-1 to G-236; M-1 to F-235; M-1 toY-234; M-1 to S-233; M-1 to R-232; M-1 to T-231; M-1 to G-230; M-1 toD-229; M-1 to R-228; M-1 to L-227; M-1 to R-226; M-1 to V-225; M-1 toL-224; M-1 to R-223; M-1 to E-222; M-1 to D-221; M-1 to L-220; M-1 toV-219; M-1 to R-218; M-1 to V-217; M-1 to V-216; M-1 to V-215; M-1 toE-214; M-1 to E-213; M-1 to G-212; M-1 to A-211; M-1 to E-210; M-1 toL-209; M-1 to H-208; M-1 to V-207; M-1 to V-206; M-1 to G-205; M-1 toG-204; M-1 to L-203; M-1 to F-202; M-1 to S-201; M-1 to S-200; M-1 toD-199; M-1 to W-198; M-1 to W-197; M-1 to V-196; M-1 to R-195; M-1 toS-194; M-1 to S-193; M-1 to S-192; M-1 to T-191; M-1 to A-190; M-1 toR-189; M-1 to G-188; M-1 to C-187; M-1 to P-186; M-1 to S-185; M-1 toQ-184; M-1 to Q-183; M-1 to S-182; M-1 to V-181; M-1 to L-180; M-1 toL-179; M-1 to E-178; M-1 to L-177; M-1 to E-176; M-1 to E-175; M-1 toP-174; M-1 to Y-173; M-1 to R-172; M-1 to P-171; M-1 to T-170; M-1 toR-169; M-1 to K-168; M-1 to Y-167; M-1 to L-166; M-1 to G-165; M-1 toH-164; M-1 to T-163; M-1 to I-162; M-1 to T-161; M-1 to S-160; M-1 toA-159; M-1 to L-158; M-1 to G-157; M-1 to L-156; M-1 to P-155; M-1 toC-154; M-1 to G-153; M-1 to V-152; M-1 to G-151; M-1 to G-150; M-1 toL-149; M-1 to Q-148; M-1 to V-147; M-1 to K-146; M-1 to S-145; M-1 toY-144; M-1 to I-143; M-1 to Y-142; M-1 to Y-141; M-1 to Y-140; M-1 toG-139; M-1 to A-138; M-1 to K-137; M-1 to T-136; M-1 to V-135; M-1 toV-134; M-1 to L-133; M-1 to A-132; M-1 to G-131; M-1 to D-130; M-1 toH-129; M-1 to Y-128; M-1 to S-127; M-1 to L-126; M-1 to G-125; M-1 toR-124; M-1 to L-123; M-1 to F-122; M-1 to A-121; M-1 to L-120; M-1 toG-119; M-1 to L-118; M-1 to Q-117; M-1 to T-116; M-1 to E-115; M-1 toW-114; M-1 to L-113; M-1 to L-112; M-1 to P-Il1; M-1 to G-110; M-1 toG-109; M-1 to S-108; M-1 to G-107; M-1 to T-106; M-1 to L-105; M-1 toS-104; M-1 to S-103; M-1 to N-102; M-1 to A-101; M-1 to G-100; M-1 toT-99; M-1 to L-98; M-1 to H-97; M-1 to A-96; M-1 to A-95; M-1 to P-94;M-1 to N-93; M-1 to V-92; M-1 to E-91; M-1 to H-90; M-1 to S-89; M-1 toR-88; M-1 to R-87; M-1 to E-86; M-1 to Q-85; M-1 to I-84; M-1 to L-83;M-1 to Q-82; M-1 to E-81; M-1 to W-80; M-1 to S-79; M-1 to G-78; M-1 toA-77; M-1 to P-76; M-1 to G-75; M-1 to D-74; M-1 to P-73; M-1 to L-72;M-1 to R-71; M-1 to T-70; M-1 to V-69; M-1 to M-68; M-1 to E-67; M-1 toG-66; M-1 to L-65; M-1 to R-64; M-1 to W-63; M-1 to H-62; M-1 to L-61;M-1 to Q-60; M-1 to L-59; M-1 to L-58; M-1 to F-57; M-1 to W-56; M-1 toG-55; M-1 to Q-54; M-1 to V-53; M-1 to A-52; M-1 to L-51; M-1 to G-50;M-1 to A-49; M-1 to G-48; M-1 to M-47; M-1 to L-46; M-1 to L-45; M-1 toL-44; M-1 to L-43; M-1 to L42; M-1 to G-41; M-1 to L-40; M-1 to G-39;M-1 to V-38; M-1 to R-37; M-1 to A-36; M-1 to V-35; M-1 to S-34; M-1 toC-33; M-1 to S-32; M-1 to Q-31; M-1 to R-30; M-1 to R-29; M-1 to H-28;M-1 to S-27; M-1 to R-26; M-1 to G-25; M-1 to L-24; M-1 to R-23; M-1 toT-22; M-1 to F-21; M-1 to P-20; M-1 to I-19; M-1 to D-18; M-1 to T-17;M-1 to Q-16; M-1 to G-15; M-1 to D-14; M-1 to V-13; M-1 to V-12; M-1 toF-11; M-1 to V-10; M-1 to S-9; M-1 to P-8; M-1 to R-7; and/or M-1 to V-6of the TL5 sequence of SEQ ID NO:2.

In another embodiment, antibodies of the invention bind C-terminaldeletions of the TL5 polypeptide that can be described by the generalformula 59-m² where m² is a number from 65 to 239 corresponding to theamino acid sequence identified of SEQ ID NO:2. In specific embodiments,the invention provides antibodies that bind TL5 polypeptides comprising,or alternatively consisting of, the amino acid sequence of residues:L-59 to M-239; L-59 to F-238; L-59 to A-237; L-59 to G-236; L-59 toF-235; L-59 to Y-234; L-59 to S-233; L-59 to R-232; L-59 to T-231; L-59to G-230; L-59 to D-229; L-59 to R-228; L-59 to L-227; L-59 to R-226;L-59 to V-225; L-59 to L-224; L-59 to R-223; L-59 to E-222; L-59 toD-221; L-59 to L-220; L-59 to V-219; L-59 to R-218; L-59 to V-217; L-59to V-216; L-59 to V-215; L-59 to E-214; L-59 to E-213; L-59 to G-212;L-59 to A-211; L-59 to E-210; L-59 to L-209; L-59 to H-208; L-59 toV-207; L-59 to V-206; L-59 to G-205; L-59 to G-204; L-59 to L-203; L-59to F-202; L-59 to S-201; L-59 to S-200; L-59 to D-199; L-59 to W-198;L-59 to W-197; L-59 to V-196; L-59 to R-195; L-59 to S-194; L-59 toS-193; L-59 to S-192; L-59 to T-191; L-59 to A-190; L-59 to R-189; L-59to G-188; L-59 to C-187; L-59 to P-186; L-59 to S-185; L-59 to Q-184;L-59 to Q-183; L-59 to S-182; L-59 to V-181; L-59 to L-180; L-59 toL-179; L-59 to E-178; L-59 to L-177; L-59 to E-176; L-59 to E-175; L-59to P-174; L-59 to Y-173; L-59 to R-172; L-59 to P-171; L-59 to T-170;L-59 to R-169; L-59 to K-168; L-59 to Y-167; L-59 to L-166; L-59 toG-165; L-59 to H-164; L-59 to T-163; L-59 to 1-162; L-59 to T-161; L-59to S-160; L-59 to A-159; L-59 to L-158; L-59 to G-157; L-59 to L-156;L-59 to P-155; L-59 to C-154; L-59 to G-153; L-59 to V-152; L-59 toG-151; L-59 to G-150; L-59 to L-149; L-59 to Q-148; L-59 to V-147; L-59to K-146; L-59 to S-145; L-59 to Y-144; L-59 to 1-143; L-59 to Y-142;L-59 to Y-141; L-59 to Y-140; L-59 to G-139; L-59 to A-138; L-59 toK-137; L-59 to T-136; L-59 to V-135; L-59 to V-134; L-59 to L-133; L-59to A-132; L-59 to G-131; L-59 to D-130; L-59 to H-129; L-59 to Y-128;L-59 to S-127; L-59 to L-126; L-59 to G-125; L-59 to R-124; L-59 toL-123; L-59 to F-122; L-59 to A-121; L-59 to L-120; L-59 to G-119; L-59to L-118; L-59 to Q-117; L-59 to T-116; L-59 to E-115; L-59 to W-114;L-59 to L-113; L-59 to L-112; L-59 to P-Ill; L-59 to G-11O; L-59 toG-109; L-59 to S-108; L-59 to G-107; L-59 to T-106; L-59 to L-105; L-59to S-104; L-59 to S-103; L-59 to N-102; L-59 to A-101; L-59 to G-100;L-59 to T-99; L-59 to L-98; L-59 to H-97; L-59 to A-96; L-59 to A-95;L-59 to P-94; L-59 to N-93; L-59 to V-92; L-59 to E-91; L-59 to H-90;L-59 to S-89; L-59 to R-88; L-59 to R-87; L-59 to E-86; L-59 to Q-85;L-59 to I-84; L-59 to L-83; L-59 to Q-82; L-59 to E-81; L-59 to W-80;L-59 to S-79; L-59 to G-78; L-59 to A-77; L-59 to P-76; L-59 to G-75;L-59 to D-74; L-59 to P-73; L-59 to L-72; L-59 to R-71; L-59 to T-70;L-59 to V-69; L-59 to M-68; L-59 to E-67; L-59 to G-66; and/or L-59 toL-65 of the TL5 sequence of SEQ ID NO:2.

The invention also provides antibodies that bind polypeptides having oneor more amino acids deleted from both the amino and the carboxyl terminiof a TL5 polypeptide, which may be described generally as havingresidues n¹-m¹ and/or n¹-m² of SEQ ID NO:2, where n¹, m¹, and m² areintegers as described above.

Preferably, antibodies of the present invention bind fragments of TL5comprising a portion of the extracellular domain; i.e., within residues59-240 of SEQ ID NO:2.

It will be recognized in the art that some amino acid sequence of TL5can be varied without significant effect of the structure or function ofthe protein. If such differences in sequence are contemplated, it shouldbe remembered that there will be critical areas on the protein whichdetermine activity. Such areas will usually comprise residues which makeup the ligand binding site or which form tertiary structures whichaffect these domains.

Thus, the invention further includes antibodies that bind variations ofthe TL5 protein which show substantial TL5 protein activity or whichinclude regions of TL5 such as the protein fragments discussed below.Such mutants include deletions, insertions, inversions, repeats, andtype substitution. Guidance concerning which amino acid changes arelikely to be phenotypically silent can be found in Bowie, J. U. et al.,Science 247:1306-1310 (1990).

Thus, antibodies of the present invention may bind a fragment,derivative, or analog of the polypeptide of SEQ ID NO:2, or that encodedby the cDNA in ATCC deposit 97689. Such fragments, variants orderivatives may be (i) one in which at least one or more of the aminoacid residues are substituted with a conserved or non-conserved aminoacid residue (preferably a conserved amino acid residue(s), and morepreferably at least one but less than ten conserved amino acid residues)and such substituted amino acid residue may or may not be one encoded bythe genetic code, or (ii) one in which one or more of the amino acidresidues includes a substituent group, or (iii) one in which the maturepolypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol), or (iv) one in which the additional amino acids are fused tothe mature polypeptide, such as an IgG Fc fusion region peptide orleader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

Of particular interest are substitutions of charged amino acids withanother charged amino acid and with neutral or negatively charged aminoacids. The latter results in proteins with reduced positive charge toimprove the characteristics of the TL5 protein. The prevention ofaggregation is highly desirable. Aggregation of proteins not onlyresults in a loss of activity but can also be problematic when preparingpharmaceutical formulations, because they can be immunogenic. (Pinckardet al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug CarrierSystems 10:307-377 (1993)).

The replacement of amino acids can also change the selectivity ofbinding to cell surface receptors. Ostade et al., Nature 361:266-268(1993) describes certain mutations resulting in selective binding ofTNF-alpha to only one of the two known types of TNF receptors. Thus, theantibodies of the present invention may bind a TL5 that contains one ormore amino acid substitutions, deletions or additions, either fromnatural mutations or human manipulation.

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table 3).

TABLE 3 Conservative Amino Acid Substitutions. Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

In specific embodiments, the number of substitutions, additions ordeletions in the amino acid sequence of SEQ ID NO:2 and/or any of thepolypeptide fragments described herein (e.g., the extracellular domainor intracellular domain) is 75, 70, 60, 50, 40, 35, 30, 25, 20, 15, 10,9, 8, 7, 6, 5, 4, 3, 2, 1 or 30-20, 20-15, 20-10, 15-10, 10-1, 5-10,1-5, 1-3 or 1-2.

In specific embodiments, the antibodies of the invention bind TL5polypeptides or fragments or variants thereof (especially a fragmentcomprising or alternatively consisting of, the extracellular solubledomain of TL5), that contains any one or more of the followingconservative mutations in TL5: M1 replaced with A, G, I, L, S, T, or V;E2 replaced with D; E3 replaced with D; S4 replaced with A, G, I, L, T,M, or V; V5 replaced with A, G, I, L, S, T, or M; V6 replaced with A, G,I, L, S, T, or M; R7 replaced with H, or K; S9 replaced with A, G, I, L,T, M, or V; V10 replaced with A, G, I, L, S, T, or M; F11 replaced withW, or Y; V12 replaced with A, G, I, L, S, T, or M; V13 replaced with A,G, I, L, S, T, or M; D14 replaced with E; G15 replaced with A, I, L, S,T, M, or V; Q16 replaced with N; T17 replaced with A, G, I, L, S, M, orV; D18 replaced with E; I19 replaced with A, G, L, S, T, M, or V; F21replaced with W, or Y; T22 replaced with A, G, I, L, S, M, or V; R23replaced with H, or K; L24 replaced with A, G, I, S, T, M, or V; G25replaced with A, I, L, S, T, M, or V; R26 replaced with H, or K; S27replaced with A, G, I, L, T, M, or V; H28 replaced with K, or R; R29replaced with H, or K; R30 replaced with H, or K; Q31 replaced with N;S32 replaced with A, G, I, L, T, M, or V; S34 replaced with A, G, I, L,T, M, or V; V35 replaced with A, G, I, L, S, T, or M; A36 replaced withG, I, L, S, T, M, or V; R37 replaced with H, or K; V38 replaced with A,G, I, L, S, T, or M; G39 replaced with A, I, L, S, T, M, or V; L40replaced with A, G, I, S, T, M, or V; G41 replaced with A, I, L, S, T,M, or V; L42 replaced with A, G, I, S, T, M, or V; L43 replaced with A,G, I, S, T, M, or V; L44 replaced with A, G, I, S, T, M, or V; L45replaced with A, G, I, S, T, M, or V; L46 replaced with A, G, I, S, T,M, or V; M47 replaced with A, G, I, L, S, T, or V; G48 replaced with A,I, L, S, T, M, or V; A49 replaced with G, I, L, S, T, M, or V; G50replaced with A, I, L, S, T, M, or V; L51 replaced with A, G, I, S, T,M, or V; A52 replaced with G, I, L, S, T, M, or V; V53 replaced with A,G, I, L, S, T, or M; Q54 replaced with N; G55 replaced with A, I, L, S,T, M, or V; W56 replaced with F, or Y; F57 replaced with W, or Y; L58replaced with A, G, I, S, T, M, or V; L59 replaced with A, G, I, S, T,M, or V; Q60 replaced with N; L61 replaced with A, G, I, S, T, M, or V;H62 replaced with K, or R; W63 replaced with F, or Y; R64 replaced withH, or K; L65 replaced with A, G, I, S, T, M, or V; G66 replaced with A,I, L, S, T, M, or V; E67 replaced with D; M68 replaced with A, G, I, L,S, T, or V; V69 replaced with A, G, I, L, S, T, or M; T70 replaced withA, G, I, L, S, M, or V; R71 replaced with H, or K; L72 replaced with A,G, I, S, T, M, or V; D74 replaced with E; G75 replaced with A, I, L, S,T, M, or V; A77 replaced with G, I, L, S, T, M, or V; G78 replaced withA, I, L, S, T, M, or V; S79 replaced with A, G, I, L, T, M, or V; W80replaced with F, or Y; E81 replaced with D; Q82 replaced with N; L83replaced with A, G, I, S, T, M, or V; 184 replaced with A, G, L, S, T,M, or V; Q85 replaced with N; E86 replaced with D; R87 replaced with H,or K; R88 replaced with H, or K; S89 replaced with A, G, I, L, T, M, orV; H90 replaced with K, or R; E91 replaced with D; V92 replaced with A,G, I, L, S, T, or M; N93 replaced with Q; A95 replaced with G, I, L, S,T, M, or V; A96 replaced with G, I, L, S, T, M, or V; H97 replaced withK, or R; L98 replaced with A, G, I, S, T, M, or V; T99 replaced with A,G, I, L, S, M, or V; G100 replaced with A, I, L, S, T, M, or V; A101replaced with G, I, L, S, T, M, or V; N102 replaced with Q; S103replaced with A, G, I, L, T, M, or V; S104 replaced with A, G, I, L, T,M, or V; L105 replaced with A, G, I, S, T, M, or V; T106 replaced withA, G, I, L, S, M, or V; G107 replaced with A, I, L, S, T, M, or V; S108replaced with A, G, I, L, T, M, or V; G109 replaced with A, I, L, S, T,M, or V; G110 replaced with A, I, L, S, T, M, or V; L112 replaced withA, G, I, S, T, M, or V; L113 replaced with A, G, I, S, T, M, or V; W114replaced with F, or Y; E115 replaced with D; T116 replaced with A, G, I,L, S, M, or V; Q117 replaced with N; L118 replaced with A, G, I, S, T,M, or V; G119 replaced with A, I, L, S, T, M, or V; L120 replaced withA, G, I, S, T, M, or V; A121 replaced with G, I, L, S, T, M, or V; F122replaced with W, or Y; L123 replaced with A, G, I, S, T, M, or V; R124replaced with H, or K; G125 replaced with A, I, L, S, T, M, or V; L126replaced with A, G, I, S, T, M, or V; S127 replaced with A, G, I, L, T,M, or V; Y128 replaced with F, or W; H129 replaced with K, or R; D130replaced with E; G131 replaced with A, I, L, S, T, M, or V; A132replaced with G, I, L, S, T, M, or V; L133 replaced with A, G, I, S, T,M, or V; V134 replaced with A, G, I, L, S, T, or M; V135 replaced withA, G, I, L, S, T, or M; T136 replaced with A, G, I, L, S, M, or V; K137replaced with H, or R; A138 replaced with G, I, L, S, T, M, or V; G139replaced with A, I, L, S, T, M, or V; Y140 replaced with F, or W; Y141replaced with F, or W; Y142 replaced with F, or W; I143 replaced with A,G, L, S, T, M, or V; Y144 replaced with F, or W; S145 replaced with A,G, I, L, T, M, or V; K146 replaced with H, or R; V147 replaced with A,G, I, L, S, T, or M; Q148 replaced with N; L149 replaced with A, G, I,S, T, M, or V; G150 replaced with A, I, L, S, T, M, or V; G151 replacedwith A, I, L, S, T, M, or V; V152 replaced with A, G, I, L, S, T, or M;G153 replaced with A, I, L, S, T, M, or V; L156 replaced with A, G, I,S, T, M, or V; G157 replaced with A, I, L, S, T, M, or V; L158 replacedwith A, G, I, S, T, M, or V; A159 replaced with G, I, L, S, T, M, or V;S160 replaced with A, G, I, L, T, M, or V; T161 replaced with A, G, I,L, S, M, or V; I162 replaced with A, G, L, S, T, M, or V; T163 replacedwith A, G, I, L, S, M, or V; H164 replaced with K, or R; G165 replacedwith A, I, L, S, T, M, or V; L166 replaced with A, G, I, S, T, M, or V;Y167 replaced with F, or W; K168 replaced with H, or R; R169 replacedwith H, or K; T170 replaced with A, G, I, L, S, M, or V; R172 replacedwith H, or K; Y173 replaced with F, or W; E175 replaced with D; E176replaced with D; L177 replaced with A, G, I, S, T, M, or V; E178replaced with D; L179 replaced with A, G, I, S, T, M, or V; L180replaced with A, G, I, S, T, M, or V; V181 replaced with A, G, I, L, S,T, or M; S182 replaced with A, G, I, L, T, M, or V; Q183 replaced withN; Q184 replaced with N; S185 replaced with A, G, I, L, T, M, or V; G188replaced with A, I, L, S, T, M, or V; R189 replaced with H, or K; A190replaced with G, I, L, S, T, M, or V; T191 replaced with A, G, I, L, S,M, or V; S192 replaced with A, G, I, L, T, M, or V; S193 replaced withA, G, I, L, T, M, or V; S194 replaced with A, G, I, L, T, M, or V; R195replaced with H, or K; V196 replaced with A, G, I, L, S, T, or M; W197replaced with F, or Y; W198 replaced with F, or Y; D199 replaced with E;S200 replaced with A, G, I, L, T, M, or V; S201 replaced with A, G, I,L, T, M, or V; F202 replaced with W, or Y; L203 replaced with A, G, I,S, T, M, or V; G204 replaced with A, I, L, S, T, M, or V; G205 replacedwith A, I, L, S, T, M, or V; V206 replaced with A, G, I, L, S, T, or M;V207 replaced with A, G, I, L, S, T, or M; H208 replaced with K, or R;L209 replaced with A, G, I, S, T, M, or V; E210 replaced with D; A211replaced with G, I, L, S, T, M, or V; G212 replaced with A, I, L, S, T,M, or V; E213 replaced with D; E214 replaced with D; V215 replaced withA, G, I, L, S, T, or M; V216 replaced with A, G, I, L, S, T, or M; V217replaced with A, G, I, L, S, T, or M; R218 replaced with H, or K; V219replaced with A, G, I, L, S, T, or M; L220 replaced with A, G, I, S, T,M, or V; D221 replaced with E; E222 replaced with D; R223 replaced withH, or K; L224 replaced with A, G, I, S, T, M, or V; V225 replaced withA, G, I, L, S, T, or M; R226 replaced with H, or K; L227 replaced withA, G, I, S, T, M, or V; R228 replaced with H, or K; D229 replaced withE; G230 replaced with A, I, L, S, T, M, or V; T231 replaced with A, G,I, L, S, M, or V; R232 replaced with H, or K; S233 replaced with A, G,I, L, T, M, or V; Y234 replaced with F, or W; F235 replaced with W, orY; G236 replaced with A, I, L, S, T, M, or V; A237 replaced with G, I,L, S, T, M, or V; F238 replaced with W, or Y; M239 replaced with A, G,I, L, S, T, or V; V240 replaced with A, G, I, L, S, T, or M of SEQ IDNO:2.

In specific embodiments, the antibodies of the invention bind TL5polypeptides or fragments or variants thereof (especially a fragmentcomprising or alternatively consisting of, the extracellular solubledomain of TL5), that contains any one or more of the followingnon-conservative mutations in TL5: M1 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; E2 replaced with H, K, R, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; E3 replaced with H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; S4 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; V5 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V6replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R7 replaced with D,E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P8 replaced with D,E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S9 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; V10 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; F11 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; V12 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; V13 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;D14 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; G15 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q16 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; T17replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D18 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I19 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; P20 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; F21 replaced with D,E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T22 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; R23 replaced with D, E, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; L24 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; G25 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; R26 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; S27 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H28replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R29replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R30replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q31replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;S32 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C33 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; S34replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V35 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; A36 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; R37 replaced with D, E, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; V38 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; G39 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L40replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G41 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; L42 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; L43 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; 44, replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L45replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L46 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; M47 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; G48 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; A49 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G50replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L51 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; A52 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; V53 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; Q54 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,Y, P, or C; G55 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W56replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F57replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L58replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L59 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; Q60 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; L61 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; H62 replaced with D, E, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; W63 replaced with D, E, H, K, R, N, Q, A, G,I, L, S, T, M, V, P, or C; R64 replaced with D, E, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; L65 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; G66 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E67replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;M68 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V69 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; T70 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; R71 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; L72 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; P73 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, or C; D74 replaced with H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; G75 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; P76 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, or C; A77 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;G78 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S79 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; W80 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; E81 replaced with H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q82 replaced with D, E,H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L83 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; 184 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; Q85 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, F, W, Y, P, or C; E86 replaced with H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; R87 replaced with D, E, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; R88 replaced with D, E, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; S89 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; H90 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; E91 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; V92 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;N93 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, orC; P94 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, or C; A95 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A96replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H97 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L98 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; T99 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; G100 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; A101 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N102replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;S103 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S104 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L105 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; T106 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; G107 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;S108 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G109 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; G110 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; P111 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or C; L112 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; L113 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; W114 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; E115 replaced with H, K, R, A, G,I, L, S, T, M, V, N, Q, F, W, Y,P, or C; T116 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q117replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;L118 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G119 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L120 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; A121 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; F122 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,M, V, P, or C; L123 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;R124 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;G125 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L126 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; S127 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; Y128 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; H129 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; D130 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; G131 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; A132 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; L133 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V134replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V135 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; T136 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; K137 replaced with D, E, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; A138 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; G139 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y140replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Y141replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Y142replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; I143replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y144 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S145 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; K146 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; V147 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; Q148 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, F, W, Y, P, or C; L149 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; G150 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;G151 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V152 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; G153 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; C154 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or P; P155 replaced with D, E, H, K, R, A,G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L156 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; G157 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; L158 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;A159 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S160 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; T161 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; I162 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; T163 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;H164 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;G165 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L166 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; Y167 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; K168 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R169 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T170 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; P171 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or C; R172 replaced with D, E, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; Y173 replaced with D, E, H, K, R, N,Q, A, G, I, L, S, T, M, V, P, or C; P174 replaced with D, E, H, K, R, A,G, I, L, S, T, M, V, N, Q, F, W, Y, or C; E175 replaced with H, K, R, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E176 replaced with H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L177 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; E178 replaced with H, K, R, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; L179 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; L180 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; V181 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S182replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q183 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; Q184 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S185replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P186 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; C187replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orP; G188 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R189replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A190replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T191 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; S192 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; S193 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; S194 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R195replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V196replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W197 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; W198 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; D199 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S200 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; S201 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; F202 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; L203 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; G204 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;G205 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V206 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; V207 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; H208 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; L209 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; E210 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, P, or C; A211 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; G212 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E213replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;E214 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; V215 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V216replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V217 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; R218 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; V219 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; L220 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; D221 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; E222 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; R223 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; L224 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;V225 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R226 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L227 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; R228 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D229 replaced with H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G230 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; T231 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; R232 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,F, W, Y, P, or C; S233 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; Y234 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, orC; F235 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, orC; G236 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A237replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F238 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; M239 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; V240 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C of SEQ ID NO:2.

Amino acids in the TL5 protein of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding or in vitro, or in vivo proliferativeactivity. Sites that are critical for ligand-receptor binding can alsobe determined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al., J. Mol.Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).In preferred embodiments, antibodies of the present invention bindregions of TL5 that are essential for TL5 function. In other preferredembodiments, antibodies of the present invention bind regions of TL5that are essential for TL5 function and inhibit or abolish TL5 function.In other preferred embodiments, antibodies of the present invention bindregions of TL5 that are essential for TL5 function and enhance TL5function.

Additionally, protein engineering may be employed to improve or alterthe characteristics of TL5 polypeptides. Recombinant DNA technologyknown to those skilled in the art can be used to create novel mutantproteins or muteins including single or multiple amino acidsubstitutions, deletions, additions or fusion proteins. Such modifiedpolypeptides can show, e.g., enhanced activity or increased stability.In addition, they may be purified in higher yields and show bettersolubility than the corresponding natural polypeptide, at least undercertain purification and storage conditions. Antibodies of the presentinvention may bind such modified TL5 polypeptides.

Non-naturally occurring variants of TL5 may be produced using art-knownmutagenesis techniques, which include, but are not limited tooligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis,site directed mutagenesis (see e.g., Carter et al., Nucl. Acids Res.13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6487 (1982)),cassette mutagenesis (see e.g., Wells et al., Gene 34:315 (1985)),restriction selection mutagenesis (see e.g., Wells et al., Philos.Trans. R. Soc. London SerA 317:415 (1986)).

Thus, the invention also encompasses antibodies that bind TL5derivatives and analogs that have one or more amino acid residuesdeleted, added, and/or substituted to generate TL5 polypeptides that arebetter suited for binding activity, therapeutic activity, expression,scale up, etc., in the host cells chosen. For example, cysteine residuescan be deleted or substituted with another amino acid residue in orderto eliminate disulfide bridges; N-linked glycosylation sites can bealtered or eliminated to achieve, for example, expression of ahomogeneous product that is more easily recovered and purified fromyeast hosts which are known to hyperglycosylate N-linked sites. To thisend, a variety of amino acid substitutions at one or both of the firstor third amino acid positions on any one or more of the glycosylationrecognition sequences in the TL5 polypeptides and/or an amino aciddeletion at the second position of any one or more such recognitionsequences will prevent glycosylation of the TL5 at the modifiedtripeptide sequence (see, e.g., Miyajimo et al., EMBO J 5(6):1193-1197).Additionally, one or more of the amino acid residues of TL5 polypeptides(e.g., arginine and lysine residues) may be deleted or substituted withanother residue to eliminate undesired processing by proteases such as,for example, furins or kexins.

The antibodies of the present invention also include antibodies thatbind a polypeptide comprising, or alternatively, consisting of thepolypeptide encoded by the deposited cDNAs (the deposit having ATCCAccession Number 97689 or 97483); a polypeptide comprising, oralternatively, consisting of the polypeptide of SEQ ID NO:2 minus theamino terminal methionine; a polypeptide comprising, or alternatively,consisting of the TL5 extracellular domain; a polypeptide comprising, oralternatively, consisting of the TL5 transmembrane domain; a polypeptidecomprising, or alternatively, consisting of the TL5 intracellulardomain; a polypeptide comprising, or alternatively, consisting ofsoluble polypeptides comprising all or part of the extracellular andintracelluar domains but lacking the transmembrane domain; as well aspolypeptides which are at least 80% identical, more preferably at least90% or 95% identical, still more preferably at least 96%, 97%, 98% or99% identical to the polypeptides described above (e.g., the polypeptideencoded by the deposited cDNA clones (the deposit having ATCC AccessionNumber 97689 or 97483), the polypeptide of SEQ ID NO:2 or SED ID NO:4,and portions of such polypeptides with at least 30 amino acids and morepreferably at least 50 amino acids.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a TL5 polypeptideis intended that the amino acid sequence of the polypeptide is identicalto the reference sequence except that the polypeptide sequence mayinclude up to five amino acid alterations per each 100 amino acids ofthe reference amino acid of the TL5 polypeptide. In other words, toobtain a polypeptide having an amino acid sequence at least 95%identical to a reference amino acid sequence, up to 5% of the amino acidresidues in the reference sequence may be deleted or substituted withanother amino acid, or a number of amino acids up to 5% of the totalamino acid residues in the reference sequence may be inserted into thereference sequence. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequence shown in SEQ ID NO:2 or SED ID NO:4 or to the amino acidsequence encoded by deposited cDNA clones can be determinedconventionally using known computer programs such the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711. When using Bestfit or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDBamino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter. According to this embodiment, if the subject sequence isshorter than the query sequence due to N- or C-terminal deletions, notbecause of internal deletions, a manual correction is made to theresults to take into consideration the fact that the FASTDB program doesnot account for N- and C-terminal truncations of the subject sequencewhen calculating global percent identity. For subject sequencestruncated at the N- and C-termini, relative to the query sequence, thepercent identity is corrected by calculating the number of residues ofthe query sequence that are N- and C-terminal of the subject sequence,which are not matched/aligned with a corresponding subject residue, as apercent of the total bases of the query sequence. A determination ofwhether a residue is matched/aligned is determined by results of theFASTDB sequence alignment. This percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thisfinal percent identity score is what is used for the purposes of thisembodiment. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence. For example, a 90 aminoacid residue subject sequence is aligned with a 100 residue querysequence to determine percent identity. The deletion occurs at theN-terminus of the subject sequence and therefore, the FASTDB alignmentdoes not show a matching/alignment of the first 10 residues at theN-terminus. The 10 unpaired residues represent 10% of the sequence(number of residues at the N- and C-termini not matched/total number ofresidues in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90residues were perfectly matched the final percent identity would be 90%.In another example, a 90 residue subject sequence is compared with a 100residue query sequence. This time the deletions are internal deletionsso there are no residues at the N- or C-termini of the subject sequencewhich are not matched/aligned with the query. In this case the percentidentity calculated by FASTDB is not manually corrected. Once again,only residue positions outside the N- and C-terminal ends of the subjectsequence, as displayed in the FASTDB alignment, which are notmatched/aligned with the query sequence are manually corrected for. Noother manual corrections are made for the purposes of this embodiment.

The present application is also directed to antibodies that bindproteins containing polypeptides at least 90%, 95%, 96%, 97%, 98% or 99%identical to the TL5 polypeptide sequence set forth herein as n¹-m¹,and/or n¹-m². In preferred embodiments, the application is directed toantibodies that bind proteins containing polypeptides at least 90%, 95%,96%, 97%, 98% or 99% identical to polypeptides having the amino acidsequence of the specific TL5 N- and C-terminal deletions recited herein.

In certain preferred embodiments, antibodies of the invention bind TL5fusion proteins as described above wherein the TL5 portion of the fusionprotein are those described as n¹-m¹, and/or n¹-m² herein.

Antibodies of the Invention may Bind Modified TL5 Polypeptides

It is specifically contemplated that antibodies of the present inventionmay bind modified forms of TL5 protein.

In specific embodiments, antibodies of the present invention bind TL5polypeptides (such as those described above) including, but not limitedto naturally purified TL5 polypeptides, TL5 polypeptides produced bychemical synthetic procedures, and TL5 polypeptides produced byrecombinant techniques from a prokaryotic or eukaryotic host, including,for example, bacterial, yeast, higher plant, insect and mammalian cellsusing, for example, the recombinant compositions and methods describedabove. Depending upon the host employed in a recombinant productionprocedure, the polypeptides may be glycosylated or non-glycosylated. Inaddition, TL5 polypeptides may also include an initial modifiedmethionine residue, in some cases as a result of host-mediatedprocesses.

In addition, TL5 proteins that antibodies of the present invention maybind can be chemically synthesized using techniques known in the art(e.g., see Creighton, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y. (1983), and Hunkapiller, et al., Nature310:105-111 (1984)). For example, a peptide corresponding to a fragmentof a TL5 polypeptide can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into the TL5polypeptide sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, omithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

The invention additionally, encompasses antibodies that bind TL5polypeptides which are differentially modified during or aftertranslation, e.g., by glycosylation, acetylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to an antibody molecule or other cellularligand, etc. Any of numerous chemical modifications may be carried outby known techniques, including but not limited to, specific chemicalcleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH₄, acetylation, formylation, oxidation, reduction,metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications to TL5 polypeptides include,for example, e.g., N-linked or O-linked carbohydrate chains, processingof N-terminal or C-terminal ends), attachment of chemical moieties tothe amino acid backbone, chemical modifications of N-linked or O-linkedcarbohydrate chains, and addition or deletion of an N-terminalmethionine residue as a result of procaryotic host cell expression. Thepolypeptides may also be modified with a detectable label, such as anenzymatic, fluorescent, isotopic or affinity label to allow fordetection and isolation of the protein.

Also provided by the invention are antibodies that bind chemicallymodified derivatives of TL5 polypeptide which may provide additionaladvantages such as increased solubility, stability and circulating timeof the polypeptide, or decreased immunogenicity (see U.S. Pat. No.4,179,337). The chemical moieties for derivitization may be selectedfrom water soluble polymers such as polyethylene glycol, ethyleneglycol/propylene glycol copolymers, carboxymethylcellulose, dextran,polyvinyl alcohol and the like. The polypeptides may be modified atrandom positions within the molecule, or at predetermined positionswithin the molecule and may include one, two, three or more attachedchemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As noted above, the polyethylene glycol may have a branched structure.Branched polyethylene glycols are described, for example, in U.S. Pat.No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72(1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999);and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosuresof each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues, glutamic acid residues and theC-terminal amino acid residue. Sulfhydryl groups may also be used as areactive group for attaching the polyethylene glycol molecules.Preferred for therapeutic purposes is attachment at an amino group, suchas attachment at the N-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins vialinkage to any of a number of amino acid residues. For example,polyethylene glycol can be linked to a proteins via covalent bonds tolysine, histidine, aspartic acid, glutamic acid, or cysteine residues.One or more reaction chemistries may be employed to attach polyethyleneglycol to specific amino acid residues (e.g., lysine, histidine,aspartic acid, glutamic acid, or cysteine) of the protein or to morethan one type of amino acid residue (e.g., lysine, histidine, asparticacid, glutamic acid, cysteine and combinations thereof) of the protein.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the proteins of the invention may beaccomplished by any number of means. For example, polyethylene glycolmay be attached to the protein either directly or by an interveninglinker. Linkerless systems for attaching polyethylene glycol to proteinsare described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys.9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998);U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO98/32466, the disclosures of each of which are incorporated herein byreference.

One system for attaching polyethylene glycol directly to amino acidresidues of proteins without an intervening linker employs tresylatedMPEG, which is produced by the modification of monmethoxy polyethyleneglycol (MPEG) using tresylchloride (ClSO₂CH₂CF₃). Upon reaction ofprotein with tresylated MPEG, polyethylene glycol is directly attachedto amine groups of the protein. Thus, the invention includesprotein-polyethylene glycol conjugates produced by reacting proteins ofthe invention with a polyethylene glycol molecule having a2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to proteins using a number ofdifferent intervening linkers. For example, U.S. Pat. No. 5,612,460, theentire disclosure of which is incorporated herein by reference,discloses urethane linkers for connecting polyethylene glycol toproteins. Protein-polyethylene glycol conjugates wherein thepolyethylene glycol is attached to the protein by a linker can also beproduced by reaction of proteins with compounds such asMPEG-succinimidylsuccinate, MPEG activated with1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. Anumber additional polyethylene glycol derivatives and reactionchemistries for attaching polyethylene glycol to proteins are describedin WO 98/32466, the entire disclosure of which is incorporated herein byreference. Pegylated protein products produced using the reactionchemistries set out herein are included within the scope of theinvention.

The number of polyethylene glycol moieties attached to each TL5polypeptide (i.e., the degree of substitution) may also vary. Forexample, the pegylated proteins of the invention may be linked, onaverage, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or morepolyethylene glycol molecules. Similarly, the average degree ofsubstitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9,8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or18-20 polyethylene glycol moieties per protein molecule. Methods fordetermining the degree of substitution are discussed, for example, inDelgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

As mentioned the antibodies of the present invention may bind TL5polypeptides that are modified by either natural processes, such asposttranslational processing, or by chemical modification techniqueswhich are well known in the art. It will be appreciated that the sametype of modification may be present in the same or varying degrees atseveral sites in a given TL5 polypeptide. TL5 polypeptides may bebranched, for example, as a result of ubiquitination, and they may becyclic, with or without branching. Cyclic, branched, and branched cyclicTL5 polypeptides may result from posttranslation natural processes ormay be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, pegylation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination. (See, for instance, PROTEINS—STRUCTUREAND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman andCompany, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OFPROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12(1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al.,Ann NY Acad Sci 663:48-62 (1992)).

Antibodies that Specifically Bind TL5

In one embodiment, the invention provides antibodies (e.g., anti-TL5antibodies comprising two heavy chains and two light chains linkedtogether by disulfide bridges) that specifically bind a TL5 polypeptide(e.g., SEQ ID NOs: 2 or 4) or fragments or variants thereof, wherein theamino acid sequence of the heavy chain and the amino acid sequence ofthe light chain are the same as the amino acid sequence of a heavy chainand a light chain of an antibody expressed by one or more cell linesreferred to in Table 1. In another embodiment, the invention providesantibodies (each consisting of two heavy chains and two light chainslinked together by disulfide bridges to form an antibody) thatspecifically bind a TL5 polypeptide (e.g., SEQ ID NOs: 2 or 4) orfragments or variants thereof, wherein the amino acid sequence of theheavy chain or the amino acid sequence of the light chain are the sameas the amino acid sequence of a heavy chain or a light chain of anantibody expressed by one or more cell lines referred to in Table 1.Specific binding to TL5 polypeptides may be determined by immunoassaysknown in the art or described herein for assaying specificantibody-antigen binding. Molecules comprising, or alternativelyconsisting of, fragments or variants of these antibodies thatspecifically bind to TL5 are also encompassed by the invention, as arenucleic acid molecules encoding these antibodies molecules, fragmentsand/or variants (e.g., SEQ ID NOs:8-10).

In one embodiment of the present invention, antibodies that specificallybind to TL5 or a fragment or variant thereof, comprise a polypeptidehaving the amino acid sequence of a heavy chain of an antibody expressedby at least one of the cell lines referred to in Table 1 and/or a lightchain of an antibody expressed by at least one of the cell linesreferred to in Table 1.

In another embodiment of the present invention, antibodies thatspecifically bind to TL5 or a fragment or variant thereof, comprise apolypeptide having the amino acid sequence of any one of the VH domainsof at least one of the scFvs referred to in Table 1 and/or any one ofthe VL domains of at least one of the scFvs referred to in Table 1. Inpreferred embodiments, antibodies of the present invention comprise theamino acid sequence of a VH domain and VL domain of the scFvs referredto in Table 1. Molecules comprising, or alternatively consisting of,antibody fragments or variants of the VH and/or VL domains of at leastone of the scFvs referred to in Table 1 that specifically bind to TL5are also encompassed by the invention, as are nucleic acid moleculesencoding these VH and VL domains, molecules, fragments and/or variants.

The present invention also provides antibodies that specifically bind toa polypeptide, or polypeptide fragment or variant of TL5, wherein saidantibodies comprise, or alternatively consist of, a polypeptide havingan amino acid sequence of any one, two, three, or more of the VH CDRscontained in a VH domain of one or more scFvs referred to in Table 1. Inparticular, the invention provides antibodies that specifically bindTL5, comprising, or alternatively consisting of, a polypeptide havingthe amino acid sequence of a VH CDR1 contained in a VH domain of one ormore scFvs referred to in Table 1. In another embodiment, antibodiesthat specifically bind TL5, comprise, or alternatively consist of, apolypeptide having the amino acid sequence of a VH CDR2 contained in aVH domain of one or more scFvs referred to in Table 1. In a preferredembodiment, antibodies that specifically bind TL5, comprise, oralternatively consist of a polypeptide having the amino acid sequence ofa VH CDR3 contained in a VH domain of one or more scFvs referred to inTable 1. Molecules comprising, or alternatively consisting of, theseantibodies, or antibody fragments or variants thereof, that specificallybind to TL5 or a TL5 fragment or variant thereof are also encompassed bythe invention, as are nucleic acid molecules encoding these antibodies,molecules, fragments and/or variants (e.g., SEQ ID NOs:8-10).

The present invention also provides antibodies that specifically bind toa polypeptide, or polypeptide fragment or variant of TL5, wherein saidantibodies comprise, or alternatively consist of, a polypeptide havingan amino acid sequence of any one, two, three, or more of the VL CDRscontained in a VL domain of one or more scFvs referred to in Table 1. Inparticular, the invention provides antibodies that specifically bindTL5, comprising, or alternatively consisting of, a polypeptide havingthe amino acid sequence of a VL CDR1 contained in a VL domain of one ormore scFvs referred to in Table 1. In another embodiment, antibodiesthat specifically bind TL5, comprise, or alternatively consist of, apolypeptide having the amino acid sequence of a VL CDR2 contained in aVL domain of one or more scFvs referred to in Table 1. In a preferredembodiment, antibodies that specifically bind TL5, comprise, oralternatively consist of a polypeptide having the amino acid sequence ofa VL CDR3 contained in a VL domain of one or more scFvs referred to inTable 1. Molecules comprising, or alternatively consisting of, theseantibodies, or antibody fragments or variants thereof, that specificallybind to TL5 or a TL5 fragment or variant thereof are also encompassed bythe invention, as are nucleic acid molecules encoding these antibodies,molecules, fragments and/or variants (e.g., SEQ ID NOs:8-10).

The present invention also provides antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants) that specifically bind to a TL5 polypeptide or polypeptidefragment or variant of TL5, wherein said antibodies comprise, oralternatively consist of, one, two, three, or more VH CDRs and one, two,three or more VL CDRs, as contained in a VH domain or VL domain of oneor more scFvs referred to in Table 1. In particular, the inventionprovides for antibodies that specifically bind to a polypeptide orpolypeptide fragment or variant of TL5, wherein said antibodiescomprise, or alternatively consist of, a VH CDR1 and a VL CDR1, a VHCDR1 and a VL CDR2, a VH CDR1 and a VL CDR3, a VH CDR2 and a VL CDR1, VHCDR2 and VL CDR2, a VH CDR2 and a VL CDR3, a VH CDR3 and a VH CDR1, a VHCDR3 and a VL CDR2, a VH CDR3 and a VL CDR3, or any combination thereof,of the VH CDRs and VL CDRs contained in a VH domain or VL domain of oneor more scFvs referred to in Table 1. In a preferred embodiment, one ormore of these combinations are from the same scFv as disclosed inTable 1. Molecules comprising, or alternatively consisting of, fragmentsor variants of these antibodies, that specifically bind to TL5 are alsoencompassed by the invention, as are nucleic acid molecules encodingthese antibodies, molecules, fragments or variants (e.g., SEQ IDNOs:8-10).

Nucleic Acid Molecules Encoding Anti-TL5 Antibodies

The present invention also provides for nucleic acid molecules,generally isolated, encoding an antibody of the invention (includingmolecules comprising, or alternatively consisting of, antibody fragmentsor variants thereof). In specific embodiments, the nucleic acidmolecules encoding an antibody of the invention comprise, oralternatively consist of SEQ ID NOs:8-10 or fragments or variantsthereof.

In a specific embodiment, a nucleic acid molecule of the inventionencodes an antibody (including molecules comprising, or alternativelyconsisting of, antibody fragments or variants thereof), comprising, oralternatively consisting of, a VH domain having an amino acid sequenceof any one of the VH domains of at least one of the scFvs referred to inTable 1 and a VL domain having an amino acid sequence of VL domain of atleast one of the scFvs referred to in Table 1. In another embodiment, anucleic acid molecule of the invention encodes an antibody (includingmolecules comprising, or alternatively consisting of, antibody fragmentsor variants thereof), comprising, or alternatively consisting of, a VHdomain having an amino acid sequence of any one of the VH domains of atleast one of the scFvs referred to in Table 1 or a VL domain having anamino acid sequence of a VL domain of at least one of the scFvs referredto in Table 1.

The present invention also provides antibodies that comprise, oralternatively consist of, variants (including derivatives) of theantibody molecules (e.g., the VH domains and/or VL domains) describedherein, which antibodies specifically bind to TL5 or fragment or variantthereof. Standard techniques known to those of skill in the art can beused to introduce mutations in the nucleotide sequence encoding amolecule of the invention, including, for example, site-directedmutagenesis and PCR-mediated mutagenesis which result in amino acidsubstitutions. Preferably, the variants (including derivatives) encodeless than 50 amino acid substitutions, less than 40 amino acidsubsitutions, less than 30 amino acid substitutions, less than 25 aminoacid substitutions, less than 20 amino acid substitutions, less than 15amino acid substitutions, less than 10 amino acid substitutions, lessthan 5 amino acid substitutions, less than 4 amino acid substitutions,less than 3 amino acid substitutions, or less than 2 amino acidsubstitutions relative to the reference VH domain, VHCDR1, VHCDR2,VHCDR3, VL domain, VLCDR1, VLCDR2, or VLCDR3. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a side chain with a similar charge. Familiesof amino acid residues having side chains with similar charges have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively,mutations can be introduced randomly along all or part of the codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for biological activity to identify mutants that retainactivity (e.g., the ability to bind TL5).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations may be silent or neutral missense mutations, i.e., have no, orlittle, effect on an antibody's ability to bind antigen. These types ofmutations may be useful to optimize codon usage, or improve ahybridoma's antibody production. Alternatively, non-neutral missensemutations may alter an antibody's ability to bind antigen. The locationof most silent and neutral missense mutations is likely to be in theframework regions, while the location of most non-neutral missensemutations is likely to be in CDR, though this is not an absoluterequirement. One of skill in the art would be able to design and testmutant molecules with desired properties such as no alteration inantigen binding activity or alteration in binding activity (e.g,improvements in antigen binding activity or change in antibodyspecificity). Following mutagenesis, the encoded protein may routinelybe expressed and the functional and/or biological activity of theencoded protein, (e.g., ability to specifically bind TL5) can bedetermined using techniques described herein or by routinely modifyingtechniques known in the art.

In a specific embodiment, an antibody of the invention (including amolecule comprising, or alternatively consisting of, an antibodyfragment or variant thereof), that specifically binds TL5 polypeptidesor fragments or variants thereof, comprises, or alternatively consistsof, an amino acid sequence encoded by a nucleotide sequence thathybridizes to a nucleotide sequence that is complementary to thatencoding one of the VH or VL domains of one or more scFvs referred to inTable 1. under stringent conditions, e.g., hybridization to filter-boundDNA in 6× sodium chloride/sodium citrate (SSC) at about 45° C. followedby one or more washes in 0.2×SSC/0.1% SDS at about 50-65° C., underhighly stringent conditions, e.g., hybridization to filter-bound nucleicacid in 6×SSC at about 45° C. followed by one or more washes in0.1×SSC/0.2% SDS at about 68° C., or under other stringent hybridizationconditions which are known to those of skill in the art (see, forexample, Ausubel, F. M. et al., eds., 1989, Current Protocols inMolecular Biology, Vol. I, Green Publishing Associates, Inc. and JohnWiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3). Nucleicacid molecules encoding these antibodies are also encompassed by theinvention.

It is well known within the art that polypeptides, or fragments orvariants thereof, with similar amino acid sequences often have similarstructure and many of the same biological activities. Thus, in oneembodiment, an antibody (including a molecule comprising, oralternatively consisting of, an antibody fragment or variant thereof),that specifically binds to a TL5 polypeptide or fragments or variants ofa TL5 polypeptide, comprises, or alternatively consists of, a VH domainhaving an amino acid sequence that is at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 99% identical, to the amino acid sequence of a VHdomain of at least one of the scFvs referred to in Table 1.

In another embodiment, an antibody (including a molecule comprising, oralternatively consisting of, an antibody fragment or variant thereof),that specifically binds to a TL5 polypeptide or fragments or variants ofa TL5 polypeptide, comprises, or alternatively consists of, a VL domainhaving an amino acid sequence that is at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 99% identical, to the amino acid sequence of a VLdomain of at least one of the scFvs referred to in Table 1.

Methods of Producing Antibodies

Antibodies in accordance with the invention are preferably preparedutilizing a phage scFv display library. Technologies utilized forachieving the same are disclosed in the patents, applications, andreferences disclosed herein.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles which carry the polynucleotide sequencesencoding them. In particular, DNA sequences encoding VH and VL domainsare amplified from animal cDNA libraries (e.g., human or murine cDNAlibraries of lymphoid tissues) or synthetic cDNA libraries. The DNAencoding the VH and VL domains are joined together by an scFv linker byPCR and cloned into a phagemid vector (e.g., p CANTAB 6 or pComb 3 HSS).The vector is electroporated in E. coli and the E. coli is infected withhelper phage. Phage used in these methods are typically filamentousphage including fd and M13 and the VH and VL domains are usuallyrecombinantly fused to either the phage gene III or gene VIII. Phageexpressing an antigen binding domain that binds to an antigen ofinterest (i.e., a TL5 polypeptide or a fragment thereof) can be selectedor identified with antigen, e.g., using labeled antigen or antigen boundor captured to a solid surface or bead. Examples of phage displaymethods that can be used to make the antibodies of the present inventioninclude, but are not limited to, those disclosed in Brinkman et al., J.Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958(1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances inImmunology 57:191-280(1994); PCT application No. PCT/GB91/01 134; PCTpublications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18719; WO 93/11236; WO 95/15982; WO 95/20401; WO97/13844; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,717; 5,780,225; 5,658,727;5,735,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

For some uses, such as for in vitro affinity maturation of an antibodyof the invention, it may be useful to express the VH and VL domains ofone or more scFvs referred to in Table 1 as single schain antibodies orFab fragments in a phage display library. For example, the cDNAsencoding the VH and VL domains of the scFvs referred to in Table 1 maybe expressed in all possible combinations using a phage display library,allowing for the selection of VIIVL combinations that bind a TL5polypeptides with preferred binding characteristics such as improvedaffinity or improved off rates. Additionally, VH and VL segments—the CDRregions of the VH and VL domains of the scFvs referred to in Table 1, inparticular, may be mutated in vitro. Expression of VH and VL domainswith “mutant” CDRs in a phage display library allows for the selectionof VH/VL combinations that bind a TL5 polypeptides with preferredbinding characteristics such as improved affinity or improved off rates.

Additional Methods of Producing Antibodies

Antibodies of the invention (including antibody fragments or variants)can be produced by any method known in the art. For example, it will beappreciated that antibodies in accordance with the present invention canbe expressed in cell lines including but not limited to myeloma celllines and hybridoma cell lines. Sequences encoding the cDNAs or genomicclones for the particular antibodies can be used for transformation of asuitable mammalian or nonmammalian host cells or to generate phagedisplay libraries, for example. Additionally, polypeptide antibodies ofthe invention may be chemically synthesized or produced through the useof recombinant expression systems.

One way to produce the antibodies of the invention would be to clone theVH and/or VL domains of the scFvs referred to in Table 1. In order toisolate the VH and VL domains from bacteria transfected with a vectorcontaining the scFv, PCR primers complementary to VH or VL nucleotidesequences (See Example 2), may be used to amplify the VH and VLsequences. The PCR products may then be cloned using vectors, forexample, which have a PCR product cloning site consisting of a 5′ and 3′single T nucleotide overhang, that is complementary to the overhangingsingle adenine nucleotide added onto the 5′ and 3′ end of PCR productsby many DNA polymerases used for PCR reactions. The VH and VL domainscan then be sequenced using conventional methods known in the art.Alternatively, the VH and VL domains may be amplified using vectorspecific primers designed to amplify the entire scFv, (i.e. the VHdomain, linker and VL domain.)

The cloned VH and VL genes may be placed into one or more suitableexpression vectors. By way of non-limiting example, PCR primersincluding VH or VL nucleotide sequences, a restriction site, and aflanking sequence to protect the restriction site may be used to amplifythe VH or VL sequences. Utilizing cloning techniques known to those ofskill in the art, the PCR amplified VH or VL domains may be cloned intovectors expressing the appropriate immunoglobulin constant region, e.g.,the human IgG1 or IgG4 constant region for VH domains, and the humankappa or lambda constant regions for kappa and lambda VL domains,respectively. Preferably, the vectors for expressing the VH or VLdomains comprise a promoter suitable to direct expression of the heavyand light chains in the chosen expression system, a secretion signal, acloning site for the immunoglobulin variable domain, immunoglobulinconstant domains, and a selection marker such as neomycin. The VH and VLdomains may also be cloned into a single vector expressing the necessaryconstant regions. The heavy chain conversion vectors and light chainconversion vectors are then co-transfected into cell lines to generatestable or transient cell lines that express full-length antibodies,e.g., IgG, using techniques known to those of skill in the art (See, forexample, Guo et al., J. Clin. Endocrinol. Metab. 82:925-31 (1997), andAmes et al., J. Immunol. Methods 184:177-86 (1995) which are hereinincorporated in their entireties by reference).

The invention provides polynucleotides comprising, or alternativelyconsisting of, a nucleotide sequence encoding an antibody of theinvention (including molecules comprising, or alternatively consistingof, antibody fragments or variants thereof). The invention alsoencompasses polynucleotides that hybridize under high stringency, oralternatively, under intermediate or lower stringency hybridizationconditions, e.g., as defined supra, to polynucleotides complementary tonucleic acids having a polynucleotide sequence that encodes an antibodyof the invention or a fragment or variant thereof.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. If the aminoacid sequences of the VH domains, VL domains and CDRs thereof, areknown, nucleotide sequences encoding these antibodies can be determinedusing methods well known in the art, i.e., the nucleotide codons knownto encode the particular amino acids are assembled in such a way togenerate a nucleic acid that encodes the antibody, of the invention.Such a polynucleotide encoding the antibody may be assembled fromchemically synthesized oligonucleotides (e.g., as described in Kutmeieret al., BioTechniques 17:242 (1994)), which, briefly, involves thesynthesis of overlapping oligonucleotides containing portions of thesequence encoding the antibody, annealing and ligating of thoseoligonucleotides, and then amplification of the ligated oligonucleotidesby PCR.

Alternatively, a polynucleotide encoding an antibody (includingmolecules comprising, or alternatively consisting of, antibody fragmentsor variants thereof) may be generated from nucleic acid from a suitablesource. If a clone containing a nucleic acid encoding a particularantibody is not available, but the sequence of the antibody molecule isknown, a nucleic acid encoding the immunoglobulin may be chemicallysynthesized or obtained from a suitable source (e.g., an antibody cDNAlibrary, or a cDNA library generated from, or nucleic acid, preferablypoly A+RNA, isolated from, any tissue or cells expressing the antibody,such as hybridoma cells or Epstein Barr virus transformed B cell linesthat express an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Once the nucleotide sequence of the antibody (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof) is determined, the nucleotide sequence of the antibodymay be manipulated using methods well known in the art for themanipulation of nucleotide sequences, e.g., recombinant DNA techniques,site directed mutagenesis, PCR, etc. (see, for example, the techniquesdescribed in Sambrook et al., 1990, Molecular Cloning, A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,John Wiley & Sons, NY, which are both incorporated by reference hereinin their entireties), to generate antibodies having a different aminoacid sequence, for example to create amino acid substitutions,deletions, and/or insertions.

In a specific embodiment, VH and VL domains of one or more scFvsreferred to in Table 1, or fragments or variants thereof, are insertedwithin antibody framework regions using recombinant DNA techniques knownin the art. In a specific embodiment, one, two, three, four, five, six,or more of the CDRs of VH and/or VL domains of one or more scFvsreferred to in Table 1, or fragments or variants thereof, is insertedwithin antibody framework regions using recombinant DNA techniques knownin the art. The antibody framework regions may be naturally occurring orconsensus antibody framework regions, and preferably human antibodyframework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479(1998) for a listing of human antibody framework regions, the contentsof which are hereby incorporated by reference in its entirety).Preferably, the polynucleotides generated by the combination of theantibody framework regions and CDRs encode an antibody (includingmolecules comprising, or alternatively consisting of, antibody fragmentsor variants thereof) that specifically binds to TL5. Preferably, asdiscussed supra, polynucleotides encoding variants of antibodies orantibody fragments having one or more amino acid substitutions may bemade within the framework regions, and, preferably, the amino acidsubstitutions do not significantly alter binding of the antibody to itsantigen. Additionally, such methods may be used to make amino acidsubstitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules, or antibody fragments or variants, lacking one ormore intrachain disulfide bonds. Other alterations to the polynucleotideare encompassed by the present invention and fall within the ordinaryskill of the art.

Xenomouse™ Technology

The ability to clone and reconstruct megabase-sized human loci in YACsand to introduce them into the mouse germline provides a powerfulapproach to elucidating the functional components of very large orcrudely mapped loci as well as generating useful models of humandisease. Furthermore, the utilization of such technology forsubstitution of mouse loci with their human equivalents could provideunique insights into the expression and regulation of human geneproducts during development, their communication with other systems, andtheir involvement in disease induction and progression.

An important practical application of such a strategy is the“humanization” of the mouse humoral immune system. Introduction of humanimmunoglobulin (Ig) loci into mice in which the endogenous Ig genes havebeen inactivated offers the opportunity to study the mechanismsunderlying programmed expression and assembly of antibodies as well astheir role in B cell development. Furthermore, such a strategy couldprovide an ideal source for production of fully human monoclonalantibodies (Mabs) an important milestone towards fulfilling the promiseof antibody therapy in human disease.

Fully human antibodies are expected to minimize the immunogenic andallergic responses intrinsic to mouse or mouse-derivatized Monoclonalantibodies and thus to increase the efficacy and safety of theadministered antibodies. The use of fully human antibodies can beexpected to provide a substantial advantage in the treatment of chronicand recurring human diseases, such as cancer, which require repeatedantibody administrations.

One approach towards this goal was to engineer mouse strains deficientin mouse antibody production with large fragments of the human Ig lociin anticipation that such mice would produce a large repertoire of humanantibodies in the absence of mouse antibodies. Large human Ig fragmentswould preserve the large variable gene diversity as well as the properregulation of antibody production and expression. By exploiting themouse machinery for antibody diversification and selection and the lackof immunological tolerance to human proteins, the reproduced humanantibody repertoire in these mouse strains should yield high affinityantibodies against any antigen of interest, including human antigens.Using the hybridoma technology, antigen-specific human Monoclonalantibodies with the desired specificity could be readily produced andselected.

This general strategy was demonstrated in connection with the generationof the first XenoMouse™ strains as published in 1994. See Green et al.Nature Genetics 7:13-21 (1994). The XenoMouse™ strains were engineeredwith yeast artificial chromosomes (YACS) containing 245 kb and 10 190kb-sized germline configuration fragments of the human heavy chain locusand kappa light chain locus, respectively, which contained core variableand constant region sequences. Id. The human Ig containing YACs provedto be compatible with the mouse system for both rearrangement andexpression of antibodies and were capable of substituting for theinactivated mouse Ig genes. This was demonstrated by their ability toinduce B-cell development, to produce an adult-like human repertoire offully human antibodies, and to generate antigen-specific humanmonoclonal antibodies. These results also suggested that introduction oflarger portions of the human Ig loci containing greater numbers of Vgenes, additional regulatory elements, and human Ig constant regionsmight recapitulate substantially the full repertoire that ischaracteristic of the human humoral response to infection andimmunization. The work of Green et al. was recently extended to theintroduction of greater than approximately 80% of the human antibodyrepertoire through introduction of megabase sized, germlineconfiguration YAC fragments of the human heavy chain loci and kappalight chain loci, respectively, to produce XenoMouse™ mice. See Mendezet al. Nature Genetics 15:146-156 (1997), Green and Jakobovits J Exp.Med. 188:483-495 (1998), Green, Journal of Immunological Methods231:11-23 (1999) and U.S. patent application Ser. No. 08/759,620, filedDec. 3, 1996, the disclosures of which are hereby incorporated byreference.

Such approach is further discussed and delineated in U.S. patentapplication Ser. No. 07/466,008, filed Jan. 12, 1990, Ser. No.07/710,515, filed Nov. 8, 1990, Ser. No. 07/919,297, filed Jul. 24,1992, Ser. No. 07/922,649, filed Jul. 30, 1992, filed Ser. No.08/031,801, filed Mar. 15,1993, Ser. No. 08/112,848, filed Aug. 27,1993, Ser. No. 08/234,145, filed Apr. 28, 1994, Ser. No. 08/376,279,filed Jan. 20, 1995, Ser. No. 08/430, 938, Apr. 27, 1995, Ser. No.0-8/464,584, filed Jun. 5, 1995, Ser. No. 08/464,582, filed Jun. 5,1995, 08/471,191, filed Jun. 5, 1995, Ser. No. 08/462,837, filed Jun. 5,1995, Ser. No. 08/486,853, filed Jun. 5, 1995, Ser. No. 08/486,857,filed Jun. 5, 1995, Ser. No. 08/486,859, filed Jun. 5, 1995, Ser. No.08/462,513, filed Jun. 5, 1995, Ser. No. 08/724,752, filed Oct. 2, 1996,and Ser. No. 08/759,620, filed Dec. 3, 1996. See also Mendez et al.Nature Genetics 15:146-156 (1997) and Green and Jakobovits J Exp. Med.188:483 495 (1998). See also European Patent No., EP 0 463 151 B1, grantpublished Jun. 12, 1996, International Patent Application No., WO94/02602, published Feb. 3, 1994, International Patent Application No.,WO 96/34096, published Oct. 31, 1996, and WO 98/24893, published Jun.11, 1998. The disclosures of each of the above-cited patents,applications, and references are hereby incorporated by reference intheir entirety.

Human anti-mouse antibody (HAMA) responses have led the industry toprepare chimeric or otherwise humanized antibodies. While chimericantibodies have a human constant region and a murine variable region, itis expected that certain human anti-chimeric antibody (HACA) responseswill be observed, particularly in chronic or multi-dose utilizations ofthe antibody. Thus, it would be desirable to provide fully humanantibodies against TL5 polypeptides in order to vitiate concerns and/oreffects of HAMA or HACA responses.

Monoclonal antibodies specific for TL5 polypeptides may be preparedusing hybridoma technology. (Kohler et al., Nature 256:495 (1975);Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J.Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies andT-Cell Hybridomas, Elsevier, N.Y., pp. 571-681 (1981)). Briefly,XenoMouse™ mice may be immunized with TL5 polypeptides. Afterimmunization, the splenocytes of such mice may be extracted and fusedwith a suitable myeloma cell line. Any suitable myeloma cell line may beemployed in accordance with the present invention; however, it ispreferable to employ the parent myeloma cell line (SP2O), available fromthe ATCC. After fusion, the resulting hybridoma cells are selectivelymaintained in HAT medium, and then cloned by limiting dilution asdescribed by Wands et al. (Gastroenterology 80:225-232 (1981)). Thehybridoma cells obtained through such a selection are then assayed toidentify clones which secrete antibodies capable of binding the TL5polypeptides.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use human or chimericantibodies. Completely human antibodies are particularly desirable fortherapeutic treatment of human patients. See also, U.S. Pat. Nos.4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50435,WO 98/24893, WO98/16654, WO 96/34096, WO 96/35735, and WO 91/10741; eachof which is incorporated herein by reference in its entirety. In aspecific embodiment, antibodies of the present invention comprise one ormore VH and VL domains of the invention and constant regions fromanother immunoglobulin molecule, preferably a human immunoglobulinmolecule. In a specific embodiment, antibodies of the present inventioncomprise one or more CDRs corresponding to the VH and VL domains of theinvention and framework regions from another immunoglobulin molecule,preferably a human immunoglobulin molecule. In other embodiments, anantibody of the present invention comprises one, two, three, four, five,six or more VL CDRs or VH CDRs corresponding to one or more of the VH orVL domains of one or more scFvs referred to in Table 1, or fragments orvariants thereof, and framework regions (and, optionally one or moreCDRs not present in the scFvs referred to in Table 1) from a humanimmunoglobulin molecule. In a preferred embodiment, an antibody of thepresent invention comprises a VH CDR3, VL CDR3, or both, correspondingto the same scFv, or different scFvs selected from the scFvs referred toin Table 1, or fragments or variants thereof, and framework regions froma human immunoglobulin.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules such asantibodies having a human variable region and a non-human (e.g., murine)immunoglobulin constant region or vice versa. Methods for producingchimeric antibodies are known in the art. See e.g., Morrison, Science229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al.,J. Immunol. Methods 125:191-202 (1989); U.S. Pat. Nos. 5,807,715;4,816,567; and 4,816,397, which are incorporated herein by reference intheir entirety. Chimeric antibodies comprising one or more CDRs fromhuman species and framework regions from a non-human immunoglobulinmolecule (e.g., framework regions from a murine, canine or felineimmunoglobulin molecule) (or vice versa) can be produced using a varietyof techniques known in the art including, for example, CDR-grafting (EP239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539;5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnickaet al., Protein Engineering 7(6):805-814 (1994); Roguska et al., PNAS91:969-973 (1994)), and chain shuffling (U.S. Patent No. 5,565,352). Ina preferred embodiment, chimeric antibodies comprise a human CDR3 havingan amino acid sequence of any one of the VH CDR3s or VL CDR3s of a VH orVL domain of one or more of the scFvs referred to in Table 1, or avariant thereof, and non-human framework regions or human frameworkregions different from those of the frameworks in the corresponding scFvdisclosed in Table 1. Often, framework residues in the framework regionswill be substituted with the corresponding residue from the CDR donorantibody to alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmannet al., Nature 352:323 (1988), which are incorporated herein byreference in their entireties.)

Intrabodies are antibodies, often scFvs, that are expressed from arecombinant nucleic acid molecule and engineered to be retainedintracellularly (e.g., retained in the cytoplasm, endoplasmic reticulum,or periplasm). Intrabodies may be used, for example, to ablate thefunction of a protein to which the intrabody binds. The expression ofintrabodies may also be regulated through the use of inducible promotersin the nucleic acid expression vector comprising the intrabody.Intrabodies of the invention can be produced using methods known in theart, such as those disclosed and reviewed in Chen et al., Hum. GeneTher. 5:595-601 (1994); Marasco, W. A., Gene Ther. 4:11-15 (1997);Rondon and Marasco, Annu. Rev. Microbiol. 51:257-283 (1997); Proba etal., J. Mol. Biol. 275:245-253 (1998); Cohen et al., Oncogene17:2445-2456 (1998); Ohage and Steipe, J. Mol. Biol. 291:1119-1128(1999); Ohage et al., J. Mol. Biol. 291:1129-1134 (1999); Wirtz andSteipe, Protein Sci. 8:2245-2250 (1999); Zhu et al., J. Immunol. Methods231:207-222 (1999); and references cited therein.

Recombinant expression of an antibody of the invention (includingantibody fragments or variants thereof (e.g., a heavy or light chain ofan antibody of the invention), requires construction of an expressionvector(s) containing a polynucleotide that encodes the antibody. Once apolynucleotide encoding an antibody molecule (e.g., a whole antibody, aheavy or light chain of an antibody, or portion thereof (preferably, butnot necessarily, containing the heavy or light chain variable domain)),of the invention has been obtained, the vector(s) for the production ofthe antibody molecule may be produced by recombinant DNA technologyusing techniques well known in the art. Thus, methods for preparing aprotein by expressing a polynucleotide containing an antibody encodingnucleotide sequence are described herein. Methods which are well knownto those skilled in the art can be used to construct expression vectorscontaining antibody coding sequences and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. The invention, thus, provides replicable vectorscomprising a nucleotide sequence encoding an antibody molecule of theinvention (e.g., a whole antibody, a heavy or light chain of anantibody, a heavy or light chain variable domain of an antibody, or aportion thereof, or a heavy or light chain CDR, a single chain Fv, orfragments or variants thereof), operably linked to a promoter. Suchvectors may include the nucleotide sequence encoding the constant regionof the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCTPublication WO 89/01036; and U.S. Pat. No. 5,122,464, the contents ofeach of which are hereby incorporated by reference in its entirety) andthe variable domain of the antibody may be cloned into such a vector forexpression of the entire heavy chain, the entire light chain, or boththe entire heavy and light chains.

The expression vector(s) is(are) transferred to a host cell byconventional techniques and the transfected cells are then cultured byconventional techniques to produce an antibody of the invention. Thus,the invention includes host cells containing polynucleotide(s) encodingan antibody of the invention (e.g., whole antibody, a heavy or lightchain thereof, or portion thereof, or a single chain antibody, or afragment or variant thereof), operably linked to a heterologouspromoter. In preferred embodiments, for the expression of entireantibody molecules, vectors encoding both the heavy and light chains maybe co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude, but are not limited to, bacteriophage particles engineered toexpress antibody fragments or variants thereof (single chainantibodies), microorganisms such as bacteria (e.g., E. coli, B.subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing antibody coding sequences;yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingantibody coding sequences; or mammalian cell systems (e.g., COS, CHO,BHK, 293, 3T3, NS0 cells) harboring recombinant expression constructscontaining promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably,bacterial cells such as Escherichia coli, and more preferably,eukaryotic cells, especially for the expression of whole recombinantantibody molecule, are used for the expression of a recombinant antibodymolecule. For example, mammalian cells such as Chinese hamster ovarycells (CHO), in conjunction with a vector such as the major intermediateearly gene promoter element from human cytomegalovirus is an effectiveexpression system for antibodies (Foecking et al., Gene 45:101 (1986);Cockett et al., Bio/Technology 8:2 (1990); Bebbington et al.,Bio/Techniques 10:169 (1992); Keen and Hale, Cytotechnology 18:207(1996)). These references are incorporated in their entireties byreference herein.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited to,the E. coli expression vector pUR278 (Ruther et al., EMBO 1. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathione5-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) may be used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. Antibody coding sequences may becloned individually into non-essential regions (for example, thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example, the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts (e.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA 8 1:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see, e.g., Bittner et al.,Methods in Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include, but are not limited to, CHO, VERY, BHK, Hela, COS, NSO,MDCK, 293, 3T3, W138, and in particular, breast cancer cell lines suchas, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammarygland cell line such as, for example, CRL7O3O and HsS78Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody may be engineered. Rather than using expression vectorswhich contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compositions that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited to,the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:8 17 (1980)) genes canbe employed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418(Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62: 191-217 (1993); TIB TECH 11(5):155-2 15 (May, 1993)); and hygro,which confers resistance to hygromycin (Santerre et al., Gene 30:147(1984)). Methods commonly known in the art of recombinant DNA technologymay be routinely applied to select the desired recombinant clone, andsuch methods are described, for example, in Ausubel et al. (eds.),Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993);Kriegler, Gene Transfer and Expression, A Laboratory Manual, StocktonPress, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds),Current Protocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, “The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells” in DNA Cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the coding sequence of theantibody, production of the antibody will also increase (Crouse et al.,Mol. Cell. Biol. 3:257 (1983)).

Vectors which use glutamine synthase (GS) or DHFR as the selectablemarkers can be amplified in the presence of the drugs methioninesulphoximine or methotrexate, respectively. An advantage of glutaminesynthase based vectors are the availabilty of cell lines (e.g., themurine myeloma cell line, NSO) which are glutamine synthase negative.Glutamine synthase expression systems can also function in glutaminesynthase expressing cells (e.g. Chinese Hamster Ovary (CHO) cells) byproviding additional inhibitor to prevent the functioning of theendogenous gene. A glutamine synthase expression system and componentsthereof are detailed in PCT publications: WO87/04462; WO86/05807;WO89/01036; WO89/10404; and WO91/06657 which are incorporated in theirentireties by reference herein. Additionally, glutamine synthaseexpression vectors that may be used according to the present inventionare commercially available from suppliers, including, for example LonzaBiologics, Inc. (Portsmouth, N.H.). Expression and production ofmonoclonal antibodies using a GS expression system in murine myelomacells is described in Bebbington et al., Bioltechnology 10:169(1992) andin Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which areincorporated in their entireties by reference herein.

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain is preferably placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc.Natl. Acad. Sci. USA 77:2 197 (1980)). The coding sequences for theheavy and light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof) has been chemically synthesized or recombinantlyexpressed, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, or more generally, a proteinmolecule, such as, for example, by chromatography (e.g., ion exchange,affinity, particularly by affinity for the specific antigen afterProtein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. Further, the antibodies of the presentinvention may be fused to heterologous polypeptide sequences describedherein or otherwise known in the art, to facilitate purification.

Antibodies of the present invention include naturally purified products,products of chemical synthetic procedures, and products produced byrecombinant techniques from a prokaryotic or eukaryotic host, including,for example, bacterial, yeast, higher plant, insect and mammalian cells.Depending upon the host employed in a recombinant production procedure,the antibodies of the present invention may be glycosylated or may benon-glycosylated. In addition, antibodies of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes.

Antibodies of the invention can be chemically synthesized usingtechniques known in the art (e.g., see Creighton, 1983, Proteins:Structures and Molecular Principles, W.H. Freeman & Co., N.Y., andHunkapiller, M., et al., 1984, Nature 310:105-111). For example, apeptide corresponding to a fragment of an antibody of the invention canbe synthesized by use of a peptide synthesizer. Furthermore, if desired,nonclassical amino acids or chemical amino acid analogs can beintroduced as a substitution or addition into the antibody polypeptidesequence. Non-classical amino acids include, but are not limited to, tothe D-isomers of the common amino acids, 2,4-diaminobutyric acid,a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid,g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,b-alanine, fluoro-amino acids, designer amino acids such as b-methylamino acids, Ca-methyl amino acids, Na-methyl amino acids, and aminoacid analogs in general. Furthermore, the amino acid can be D(dextrorotary) or L (levorotary).

The invention encompasses antibodies which are differentially modifiedduring or after translation, e.g., by glycosylation, acetylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to an antibody molecule or othercellular ligand, etc. Any of numerous chemical modifications may becarried out by known techniques, including but not limited, to specificchemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH4, acetylation, formylation, oxidation, reduction,metabolic synthesis in the presence of tunicamycin, etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The antibodies may also be modified with a detectable label,such as an enzymatic, fluorescent, radioisotopic or affinity label toallow for detection and isolation of the antibody.

Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, beta-galactosidase, glucose oxidase oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include biotin, umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride or phycoerythrin; an example of aluminescent material includes luminol; examples of bioluminescentmaterials include luciferase, luciferin, and aequorin; and examples ofsuitable radioactive material include a radioactive metal ion, e.g.,alpha-emitters such as, for example, ²¹³Bi, or other radioisotopes suchas, for example, iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur(³⁵S), tritium (³H), indium (^(115m)In, ^(113m)In, ¹¹²In, ¹¹¹In), andtechnetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga),palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³ Xe), fluorine (¹⁸F),¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re,¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb,⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and ¹¹⁷Tin.

In specific embodiments, antibodies of the invention may be labeled withEuropium. For example, antibodies of the invention may be labeled withEuropium using the DELFIA Eu-labeling kit (catalog #1244-302, PerkinElmer Life Sciences, Boston, Mass.) following manufacturer'sinstructions.

In specific embodiments, antibodies of the invention are attached tomacrocyclic chelators useful for conjugating radiometal ions, includingbut not limited to, ¹¹¹In, ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, ¹⁵³Sm, ²¹⁵Bi and ²²⁵Ac topolypeptides. In a preferred embodiment, the radiometal ion associatedwith the macrocyclic chelators attached to antibodies of the inventionis ¹¹¹In. In another preferred embodiment, the radiometal ion associatedwith the macrocyclic chelator attached to antibodies polypeptides of theinvention is ⁹⁰Y. In specific embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). Inspecific embodiments, the macrocyclic chelator isα-(5-isothiocyanato-2-methoxyphenyl)-1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraaceticacid. In other specific embodiments, the DOTA is attached to theantibody of the invention via a linker molecule. Examples of linkermolecules useful for conjugatinga macrocyclic chelator such as DOTA to apolypeptide are commonly known in the art—see, for example, DeNardo etal., Clin Cancer Res. 4(10):2483-90, 1998; Peterson et al., Bioconjug.Chem. 10(4):553-7, 1999; and Zimmerman et al, Nucl. Med. Biol.26(8):943-50, 1999 which are hereby incorporated by reference in theirentirety. In addition, U.S. Pat. Nos. 5,652,361 and 5,756,065, whichdisclose chelating agents that may be conjugated to antibodies, andmethods for making and using them, are hereby incorporated by referencein their entireties.

In one embodiment, antibodies of the invention are labeled with biotin.In other related embodiments, biotinylated antibodies of the inventionmay be used, for example, as an imaging agent or as a means ofidentifying one or more TRAIL receptor coreceptor or ligand molecules.

Also provided by the invention are chemically modified derivatives ofantibodies of the invention which may provide additional advantages suchas increased solubility, stability and in vivo or in vitro circulatingtime of the polypeptide, or decreased immunogenicity (see U.S. Pat. No.4,179,337). The chemical moieties for derivitization may be selectedfrom water soluble polymers such as polyethylene glycol, ethyleneglycol/propylene glycol copolymers, carboxymethylcellulose, dextran,polyvinyl alcohol and the like. The antibodies may be modified at randompositions within the molecule, or at predetermined positions within themolecule and may include one, two, three or more attached chemicalmoieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As noted above, the polyethylene glycol may have a branched structure.Branched polyethylene glycols are described, for example, in U.S. Pat.No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72(1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999);and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosuresof each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the antibody with consideration of effects on functional orantigenic domains of the antibody. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include, for example, lysineresidues and the N-terminal amino acid residues; those having a freecarboxyl group may include aspartic acid residues, glutamic acidresidues, and the C-terminal amino acid residue. Sulfhydryl groups mayalso be used as a reactive group for attaching the polyethylene glycolmolecules. Preferred for therapeutic purposes is attachment at an aminogroup, such as attachment at the N-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins,e.g., antibodies, via linkage to any of a number of amino acid residues.For example, polyethylene glycol can be linked to a proteins viacovalent bonds to lysine, histidine, aspartic acid, glutamic acid, orcysteine residues. One or more reaction chemistries may be employed toattach polyethylene glycol to specific amino acid residues (e.g.,lysine, histidine, aspartic acid, glutamic acid, or cysteine) of theprotein or to more than one type of amino acid residue (e.g., lysine,histidine, aspartic acid, glutamic acid, cysteine and combinationsthereof) of the protein.

One may specifically desire antibodies chemically modified at theN-terminus of either the heavy chain or the light chain or both. Usingpolyethylene glycol as an illustration, one may select from a variety ofpolyethylene glycol molecules (by molecular weight, branching, etc.),the proportion of polyethylene glycol molecules to protein (or peptide)molecules in the reaction mix, the type of pegylation reaction to beperformed, and the method of obtaining the selected N-terminallypegylated protein. The method of obtaining the N-terminally pegylatedpreparation (i.e., separating this moiety from other monopegylatedmoieties if necessary) may be by purification of the N-terminallypegylated material from a population of pegylated protein molecules.Selective chemical modification at the N-terminus may be accomplished byreductive alkylation which exploits differential reactivity of differenttypes of primary amino groups (lysine versus the N-terminal) availablefor derivatization in a particular protein. Under the appropriatereaction conditions, substantially selective derivatization of theprotein at the N-terminus with a carbonyl group containing polymer isachieved.

As indicated above, pegylation of the antibodies of the invention may beaccomplished by any number of means. For example, polyethylene glycolmay be attached to the antibody either directly or by an interveninglinker. Linkerless systems for attaching polyethylene glycol to proteinsare described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys.9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998);U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO98/32466, the disclosures of each of which are incorporated herein byreference.

One system for attaching polyethylene glycol directly to amino acidresidues of antibodies without an intervening linker employs tresylatedMPEG, which is produced by the modification of monmethoxy polyethyleneglycol (MPEG) using tresylchloride (ClSO2CH2CF3). Upon reaction ofprotein with tresylated MPEG, polyethylene glycol is directly attachedto amine groups of the protein. Thus, the invention includesantibody-polyethylene glycol conjugates produced by reacting antibodiesof the invention with a polyethylene glycol molecule having a2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to antibodies using a number ofdifferent intervening linkers. For example, U.S. Pat. No. 5,612,460, theentire disclosure of which is incorporated herein by reference,discloses urethane linkers for connecting polyethylene glycol toproteins. Antibody-polyethylene glycol conjugates wherein thepolyethylene glycol is attached to the antibody by a linker can also beproduced by reaction of antibodies with compounds such asMPEG-succinimnidylsuccinate, MPEG activated with1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. Anumber additional polyethylene glycol derivatives and reactionchemistries for attaching polyethylene glycol to proteins are describedin WO 98/32466, the entire disclosure of which is incorporated herein byreference. Pegylated antibody products produced using the reactionchemistries set out herein are included within the scope of theinvention.

The number of polyethylene glycol moieties attached to each antibody ofthe invention (i.e., the degree of substitution) may also vary. Forexample, the pegylated antibodies of the invention may be linked, onaverage, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or morepolyethylene glycol molecules. Similarly, the average degree ofsubstitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9,8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or18-20 polyethylene glycol moieties per antibody molecule. Methods fordetermining the degree of substitution are discussed, for example, inDelgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

Characterization of Anti-TL5 Antibodies

Antibodies of the present invention (including molecules comprising, oralternatively consisting of, antibody fragments or variants thereof) mayalso be described or specified in terms of their binding to TL5polypeptides or fragments or variants of TL5 polypeptides. In specificembodiments, antibodies of the invention bind TL5 polypeptides, orfragments or variants thereof, with a dissociation constant or K_(D) ofless than or equal to 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴M, 5×10⁻⁵ M, or 10⁻⁵ M. More preferably, antibodies of the inventionbind TL5 polypeptides or fragments or variants thereof with adissociation constant or K_(D) less than or equal to 5×10⁻⁶ M, 10⁻⁶ M,5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, or 10⁻⁸ M. Even more preferably, antibodiesof the invention bind TL5 polypeptides or fragments or variants thereofwith a dissociation constant or K_(D) less than or equal to 5×10⁻⁹ M,10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M,5×⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M. Theinvention encompasses antibodies that bind TL5 polypeptides with adissociation constant or K_(D) that is within any one of the ranges thatare between each of the individual recited values.

In specific embodiments, antibodies of the invention bind TL5polypeptides or fragments or variants thereof with an off rate (k_(off))of less than or equal to 5×10⁻² sec⁻¹, 10⁻² sec⁻¹, 5×10⁻³ sec⁻¹ or 10⁻³sec⁻¹. More preferably, antibodies of the invention bind TL5polypeptides or fragments or variants thereof with an off rate (k_(off))less than or equal to 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵sec⁻¹ 5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹ or 10⁻⁷ sec⁻¹. Theinvention encompasses antibodies that bind TL5 polypeptides with an offrate (k_(off)) that is within any one of the ranges that are betweeneach of the individual recited values.

In other embodiments, antibodies of the invention bind TL5 polypeptidesor fragments or variants thereof with an on rate (k_(on)) of greaterthan or equal to 10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹ or 5×10⁴M⁻¹ sec⁻¹. More preferably, antibodies of the invention bind TL5polypeptides or fragments or variants thereof with an on rate (k_(on))greater than or equal to 10⁵ M⁻¹ sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹,or 5×10⁶ M⁻¹ sec⁻¹ or 10⁷ M⁻¹ sec⁻¹. The invention encompassesantibodies that bind TL5 polypeptides with on rate (k_(on)) that iswithin any one of the ranges that are between each of the individualrecited values.

The antibodies of the invention (including molecules comprising, oralternatively consisting of, antibody fragments or variants thereof)specifically bind to a polypeptide or polypeptide fragment or variant ofhuman TL5 polypeptides (SEQ ID NOS:2 or 4). In another embodiment, theantibodies of the invention specifically bind to a polypeptide orpolypeptide fragment or variant of simian TL5 polypeptides. In yetanother embodiment, the antibodies of the invention specifically bind toa polypeptide or polypeptide fragment or variant of murine TL5polypeptides. In one embodiment, the antibodies of the invention bindspecifically to human and simian TL5 polypeptides. In anotherembodiment, the antibodies of the invention bind specifically to humanTL5 polypeptides and murine TL5 polypeptides. More preferably,antibodies of the invention, preferentially bind to human TL5polypeptides compared to murine TL5 polypeptides.

In preferred embodiments, the antibodies of the present invention(including molecules comprising, or alternatively consisting of,antibody fragments or variants thereof), specifically bind to TL5polypeptides and do not cross-react with any other antigens. Inpreferred embodiments, the antibodies of the invention specifically bindto TL5 polypeptides (e.g., SEQ ID NOS:2, 4 or fragments or variantsthereof) and do not cross-react with one or more additional members ofthe Tumor Necrosis Factor Tumor Necrosis Factor Family polypeptides(e.g., TNFα, TNFβ (lymphotoxin-α), LTβ, OX40L, Fas ligand, CD30L, CD27L,CD40L and 4-IBBL).

In another embodiment, the antibodies of the present invention(including molecules comprising, or alternatively consisting of,antibody fragments or variants thereof), specifically bind to TL5polypeptides and cross-react with other antigens. In other embodiments,the antibodies of the invention specifically bind to TL5 polypeptides(e.g., SEQ ID NOS:2, 4, or fragments or variants thereof) andcross-react with one or more additional members of the Tumor NecrosisFactor Family polypeptides (e.g., TNFα, TNFβ (lymphotoxin-α, LTβ, OX40L,Fas ligand, CD30L, CD27L, CD40L and 4-IBBL).

By way of non-limiting example, an antibody may be considered to bind afirst antigen preferentially if it binds said first antigen with adissociation constant (K_(D)) that is less than the antibody's K_(D) forthe second antigen. In another non-limiting embodiment, an antibody maybe considered to bind a first antigen preferentially if it binds saidfirst antigen with an affinity (i.e., K_(D)) that is at least one orderof magnitude less than the antibody's K_(D) for the second antigen. Inanother non-limiting embodiment, an antibody may be considered to bind afirst antigen preferentially if it binds said first antigen with anaffinity (i.e., K_(D)) that is at least two orders of magnitude lessthan the antibody's K_(D) for the second antigen.

In another non-limiting embodiment, an antibody may be considered tobind a first antigen preferentially if it binds said first antigen withan off rate (k_(off)) that is less than the antibody's k_(off) for thesecond antigen. In another non-limiting embodiment, an antibody may beconsidered to bind a first antigen preferentially if it binds said firstantigen with a k_(off) that is at least one order of magnitude less thanthe antibody's k_(off) for the second antigen. In another non-limitingembodiment, an antibody may be considered to bind a first antigenpreferentially if it binds said first antigen with a k_(off) that is atleast two orders of magnitude less than the antibody's k_(off) for thesecond antigen.

The invention also encompasses antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof) that have one or more of the same biologicalcharacteristics as one or more of the antibodies described herein. By“biological characteristics” is meant, the in vitro or in vivoactivities or properties of the antibodies, such as, for example, theability to bind to TL5 polypeptides (e.g., TL5 expressed on the surfaceof a cell), the ability to inhibit TL5 mediated biological activity(e.g., the ability to inhibit or abolish TL5 costimulatory activity on Tcells, see Examples 4 and 5, or the ability to inhibit proliferationand/or differentiation of TL5 and/or TL5 receptor expressing cells,especially TR2 expressing cells); or the ability, to substantially blockbinding of TL5, or a fragment, variant or fusion protein thereof, to itsreceptor (e.g. TR6 (described in WO 98/30694, WO2000/52028,WO2002/18622, and SEQ ID NO:49), LTβR (See, Genbank™ Accession NumbersL04270 and P36941 and SEQ ID NO:47), and TR2 (described in WO 96/34095,WO98/18824, WO00/56405 and SEQ ID NO:48); or the ability to inhibitapoptosis of TL5 receptor expressing cells, especially LTβR expressingcells. Other biological activities that antibodies against TL5polypeptides may have, include, but are not limited to, the ability tostimulate TL5 mediated biological activity (e.g., to stimulate TL5costimulatory activity on T cells, to stimulate proliferation and/ordifferentiation of TL5 expressing cells (e.g., T cells)) or the abilityto stimulate apoptosis of TL5 receptor expressing cells, especially LTβRexpressing cells. Optionally, the antibodies of the invention will bindto the same epitope as at least one of the antibodies specificallyreferred to herein. Such epitope binding can be routinely determinedusing assays known in the art.

The present invention also provides for antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof), that inhibit one or more TL5 polypeptide mediatedbiological activities. In one embodiment, an antibody that inhibits oneor more TL5 polypeptide mediated biological activities comprises, oralternatively consists of a VH and/or a VL domain of at least one of thescFvs referred to in Table 1, or fragment or variant thereof. In aspecific embodiment, an antibody that inhibits one or more TL5polypeptide mediated biological activities comprises, or alternativelyconsists of a VH and a VL domain of any one of the scFvs referred to inTable 1, or fragment or variant thereof. Nucleic acid molecules encodingthese antibodies are also encompassed by the invention.

The present invention also provides for antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof), that inhibit proliferation of TL5 and/or TL5 receptorexpressing cells, especially cells expressing TR2. In one embodiment, anantibody that inhibits proliferation of TL5 and/or TL5 receptorexpressing cells comprises, or alternatively consists of a VH and/or aVL domain of at least one of the scFvs referred to in Table 1, orfragment or variant thereof. In a specific embodiment, an antibody thatinhibits proliferation of TL5 and/or TL5 receptor expressing cellscomprises, or alternatively consists of a VH and a VL domain of any oneof the scFvs referred to in Table 1, or fragment or variant thereof.Nucleic acid molecules encoding these antibodies are also encompassed bythe invention.

The present invention also provides for antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof), that inhibit differentiation of TL5 and/or TL5receptor expressing cells (e.g., T cells; See Example 4 and 5). In oneembodiment, an antibody that inhibits differentiation of TL5 and/or TL5receptor expressing cells comprises, or alternatively consists of a VHand/or a VL domain of at least one of the scFvs referred to in Table 1,or fragment or variant thereof. In a specific embodiment, an antibodythat inhibits differentiation of TL5 expressing cells comprises, oralternatively consists of a VH and a VL domain of any one of the scFvsreferred to in Table 1, or fragment or variant thereof. Nucleic acidmolecules encoding these antibodies are also encompassed by theinvention.

The present invention also provides for antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof), that inhibit apoptosis of TL5 and/or TL5 receptorexpressing cells, especially LTβR expressing cells. In one embodiment,an antibody that inhibits apoptosis of TL5 and/or TL5 receptorexpressing cells comprises, or alternatively consists of a VH and/or aVL domain of at least one of the scFvs referred to in Table 1, orfragment or variant thereof. In a specific embodiment, an antibody thatinhibits apoptosis of TL5 and/or TL5 receptor expressing cellscomprises, or alternatively consists of a VH and a VL domain of any oneof the scFvs referred to in Table 1, or fragment or variant thereof.Nucleic acid molecules encoding these antibodies are also encompassed bythe invention.

The present invention also provides for antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof), that block or inhibit the binding of TL5 ligand to aTL5 receptor. In one embodiment, an antibody that blocks or inhibits thebinding of TL5 ligand to a TL5 receptor comprises, or alternativelyconsists of a VH and/or a VL domain of at least one of the scFvsreferred to in Table 1, or fragment or variant thereof. In a specificembodiment, an antibody that blocks or inhibits the binding of TL5ligand to a TL5 receptor comprises, or alternatively consists of a VHand a VL domain of any one of the scFvs referred to in Table 1, orfragment or variant thereof. In a specific embodiment, the TL5 receptoris TR2. In another specific embodiment the TL5 receptor is TR6. Inanother specific embodiment the TL5 receptor is LTβR. Nucleic acidmolecules encoding these antibodies are also encompassed by theinvention.

The present invention also provides for antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof), that stimulate proliferation and/or differentiationof TL5 and/or TL5 receptor expressing cells (e.g., T cells). In oneembodiment, an antibody that stimulates proliferation and/ordifferentiation of TL5 and/or TL5 receptor expressing cells comprises,or alternatively consists of a VH and/or a VL domain of at least one ofthe scFvs referred to in Table 1, or fragment or variant thereof. In aspecific embodiment, an antibody that stimulates proliferation and/ordifferentiation of TL5 and/or TL5 receptor expressing cells comprises,or alternatively consists of a VH and a VL domain of any one of thescFvs referred to in Table 1, or fragment or variant thereof. Nucleicacid molecules encoding these antibodies are also encompassed by theinvention.

Antibodies of the present invention (including antibody fragments orvariants thereof) may be characterized in a variety of ways. Inparticular, antibodies and related molecules of the invention may beassayed for the ability to specifically bind to TL5 polypeptides or afragment or variant of TL5 polypeptides using techniques describedherein or routinely modifying techniques known in the art. Assays forthe ability of the antibodies of the invention to specifically bind TL5polypeptides or a fragment of TL5 polypeptides may be performed insolution (e.g., Houghten, Bio/Techniques 13:412-421(1992)), on beads(e.g., Lam, Nature 354:82-84 (1991)), on chips (e.g., Fodor, Nature364:555-556 (1993)), on bacteria (e.g., U.S. Pat. No. 5,223,409), onspores (e.g., U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), onplasmids (e.g., Cull et al., Proc. Natl. Acad. Sci. USA 89:1865-1869(1992)) or on phage (e.g., Scott and Smith, Science 249:386-390 (1990);Devlin, Science 249:404-406 (1990); Cwirla et al., Proc. Natl. Acad.Sci. USA 87:7178-7182 (1990); and Felici, J. Mol. Biol. 222:301-310(1991)) (each of these references is incorporated herein in its entiretyby reference). Antibodies that have been identified to specifically bindto TL5 polypeptides or a fragment or variant of TL5 polypeptides canthen be assayed for their specificity and affinity for TL5 polypeptidesor a fragment of TL5 polypeptides using or routinely modifyingtechniques described herein or otherwise known in the art.

The antibodies of the invention may be assayed for specific binding toTL5 polypeptides and cross-reactivity with other antigens by any methodknown in the art. Immunoassays which can be used to analyze specificbinding and cross-reactivity include, but are not limited to,competitive and non-competitive assay systems using techniques such asBIAcore analysis, FACS (fluorescence activated cell sorter) analysis,immunofluorescence, immunocytochemistry, radioimmunoassays, ELISA(enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, western blots, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, and protein A immunoassays, to name but a few.Such assays are routine and well known in the art (see, e.g., Ausubel etal., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York, which is incorporated by reference hereinin its entirety). Exemplary immunoassays are described briefly below(but are not intended by way of limitation).

ELISAs comprise preparing antigen, coating the well of a 96-wellmicrotiter plate with the antigen, washing away antigen that did notbind the wells, adding the antibody of interest conjugated to adetectable compound such as an enzymatic substrate (e.g., horseradishperoxidase or alkaline phosphatase) to the wells and incubating for aperiod of time, washing away unbound antibodies or non-specificallybound antibodies, and detecting the presence of the antibodiesspecifically bound to the antigen coating the well. In ELISAs, theantibody of interest does not have to be conjugated to a detectablecompound; instead, a second antibody (which recognizes the antibody ofinterest) conjugated to a detectable compound may be added to the well.Alternatively, the antigen need not be directly coated to the well;instead the ELISA plates may be coated with an anti-Ig Fc antibody, andthe antigen in the form of a TL5-Fc fusion protein, may be bound to theanti-Ig Fc coated to the plate. This may be desirable so as to maintainthe antigen protein (e.g., the TL5 polypeptides) in a more nativeconformation than it may have when it is directly coated to a plate. Inanother alternative, instead of coating the well with the antigen, theantibody may be coated to the well. In this case, the detectablemolecule could be the antigen conjugated to a detectable compound suchas an enzymatic substrate (e.g., horseradish peroxidase or alkalinephosphatase). One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected as wellas other variations of ELISAs known in the art. For further discussionregarding ELISAs see, e.g., Ausubel et al., eds, 1994, Current Protocolsin Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at11.2.1.

The binding affinity of an antibody (including an scFv or other moleculecomprising, or alternatively consisting of, antibody fragments orvariants thereof) to an antigen and the off-rate of an antibody-antigeninteraction can be determined by competitive binding assays. One exampleof a competitive binding assay is a radioimmunoassay comprising theincubation of labeled antigen (e.g., antigen labeled with ³H or ¹²⁵I),or fragment or variant thereof with the antibody of interest in thepresence of increasing amounts of unlabeled antigen, and the detectionof the antibody bound to the labeled antigen. The affinity of theantibody of the present invention for TL5 and the binding off-rates canbe determined from the data by Scatchard plot analysis. Competition witha second antibody can also be determined using radioimmunoassays. Inthis case, TL5 polypeptide is incubated with an antibody of the presentinvention conjugated to a labeled compound (e.g., compound labeled with³H or ¹²⁵I) in the presence of increasing amounts of an unlabeled secondanti-TL5 antibody. This kind of competitive assay between twoantibodies, may also be used to determine if two antibodies bind thesame closely associated (e.g., overlapping) or different epitopes.

In a preferred embodiment, BIAcore kinetic analysis is used to determinethe binding on and off rates of antibodies (including antibody fragmentsor variants thereof) to TL5, or fragments of TL5. BIAcore kineticanalysis comprises analyzing the binding and dissociation of antibodiesfrom chips with immobilized TL5 on their surface.

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1 to 4 hours) at 40 degrees C., adding protein Aand/or protein G sepharose beads to the cell lysate, incubating forabout an hour or more at 40 degrees C., washing the beads in lysisbuffer and resuspending the beads in SDS/sample buffer. The ability ofthe antibody of interest to immunoprecipitate a particular antigen canbe assessed by, e.g., western blot analysis. One of skill in the artwould be knowledgeable as to the parameters that can be modified toincrease the binding of the antibody to an antigen and decrease thebackground (e.g., pre-clearing the cell lysate with sepharose beads).For further discussion regarding immunoprecipitation protocols see,e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., ³²P or ¹²⁵I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

Antibody Conjugates

The present invention encompasses antibodies (including antibodyfragments or variants thereof), recombinantly fused or chemicallyconjugated (including both covalent and non-covalent conjugations) to aheterologous polypeptide (or portion thereof, preferably at least 10, atleast 20, at least 30, at least 40, at least 50, at least 60, at least70, at least 80, at least 90 or at least 100 amino acids of thepolypeptide) to generate fusion proteins. The fusion does notnecessarily need to be direct, but may occur through linker sequences.For example, antibodies of the invention may be used to targetheterologous polypeptides to particular cell types (e.g., cancer cells),either in vitro or in vivo, by fusing or conjugating the heterologouspolypeptides to antibodies of the invention that are specific forparticular cell surface antigens or which bind antigens that bindparticular cell surface receptors. Antibodies of the invention may alsobe fused to albumin (including but not limited to recombinant humanserum albumin (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999,EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998,herein incorporated by reference in their entirety)), resulting inchimeric polypeptides. In a preferred embodiment, polypeptides and/orantibodies of the present invention (including fragments or variantsthereof) are fused with the mature form of human serum albumin (i.e.,amino acids 1-585 of human serum albumin as shown in FIGS. 1 and 2 of EPPatent 0 322 094) which is herein incorporated by reference in itsentirety. In another preferred embodiment, polypeptides and/orantibodies of the present invention (including fragments or variantsthereof) are fused with polypeptide fragments comprising, oralternatively consisting of, amino acid residues 1-z of human serumalbumin, where z is an integer from 369 to 419, as described in U.S.Pat. No. 5,766,883 herein incorporated by reference in its entirety.Polypeptides and/or antibodies of the present invention (includingfragments or variants thereof) may be fused to either the N- orC-terminal end of the heterologous protein (e.g., immunoglobulin Fcpolypeptide or human serum albumin polypeptide). Polynucleotidesencoding fusion proteins of the invention are also encompassed by theinvention. Such fusion proteins may, for example, facilitatepurification and may increase half-life in vivo. Antibodies fused orconjugated to heterologous polypeptides may also be used in in vitroimmunoassays and purification methods using methods known in the art.See e.g., Harbor et al., supra, and PCT publication WO 93/2 1232; EP439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No.5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J.Immunol. 146:2446-2452 (1991), which are incorporated by reference intheir entireties.

The present invention further includes compositions comprising, oralternatively consisting of, heterologous polypeptides fused orconjugated to antibody fragments. For example, the heterologouspolypeptides may be fused or conjugated to a Fab fragment, Fd fragment,Fv fragment, F(ab)₂ fragment, or a portion thereof. Methods for fusingor conjugating polypeptides to antibody portions are known in the art.See, e.g., U.S. Pat. Nos. 5,356,603; 5,622,929; 5,359,046; 5,349,053;5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO96/04388; WO 9 1/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995); andVil et al., Proc. Natl. Acad. Sci. USA 89:11357-11341 (1992) (saidreferences incorporated by reference in their entireties).

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of antibodies(including molecules comprising, or alternatively consisting of,antibody fragments or variants thereof), such methods can be used togenerate antibodies with altered activity (e.g., antibodies with higheraffinities and lower dissociation rates). See, generally, U.S. Pat. Nos.5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten etal., Curr. Opinion Biotechnol. 8:724-35 (1997); Harayama, TrendsBiotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol.287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13(1998) (each of these patents and publications are hereby incorporatedby reference in its entirety). In one embodiment, polynucleotidesencoding antibodies of the invention may be altered by being subjectedto random mutagenesis by error-prone PCR, random nucleotide insertion orother methods prior to recombination. In another embodiment, one or moreportions of a polynucleotide encoding an antibody which portionsspecifically bind to TL5 may be recombined with one or more components,motifs, sections, parts, domains, fragments, etc. of one or moreheterologous molecules.

Moreover, the antibodies of the present invention (including antibodyfragments or variants thereof), can be fused to marker sequences, suchas a polypeptides to facilitate purification. In preferred embodiments,the marker amino acid sequence is a hexa-histidine polypeptide, such asthe tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,Chatsworth, Calif., 91311), among others, many of which are commerciallyavailable. As described in Gentz et al., Proc. Natl. Acad. Sci. USA86:821-824 (1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. Other peptide tags useful forpurification include, but are not limited to, the hemagglutinin “HA”tag, which corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the FLAG®tag (Stratagene, La Jolla, Calif.).

The present invention further encompasses antibodies (including antibodyfragments or variants thereof), conjugated to a diagnostic ortherapeutic agent. The antibodies can be used diagnostically to, forexample, monitor or prognose the development or progression of a tumoras part of a clinical testing procedure to, e.g., determine the efficacyof a given treatment regimen. Detection can be facilitated by couplingthe antibody to a detectable substance. Examples of detectablesubstances include, but are not limited to, various enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, radioactive materials, positron emitting metals using variouspositron emission tomographies, and nonradioactive paramagnetic metalions. The detectable substance may be coupled or conjugated eitherdirectly to the antibody or indirectly, through an intermediate (suchas, for example, a linker known in the art) using techniques known inthe art. See, for example, U.S. Pat. No. 4,741,900 for metal ions whichcan be conjugated to antibodies for use as diagnostics according to thepresent invention. Examples of suitable enzymes include, but are notlimited to, horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include, but are not limited to,streptavidin/biotin and avidinibiotin; examples of suitable fluorescentmaterials include, but are not limited to, umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride or phycoerythrin; an example of aluminescent material includes, but is not limited to, luminol; examplesof bioluminescent materials include, but are not limited to, luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude, but are not limited to, iodine (¹²¹I, ¹²³I, ¹²³I, ¹³¹I), carbon(¹⁴C), sulfur (35S), tritium (³H), indium (¹¹¹In, ¹¹²In, ^(113m)In,^(115m)In), technetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium(⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³⁵Xe),fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y,⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, and ⁹⁷Ru.

Further, an antibody of the invention (including an scFv or othermolecule comprising, or alternatively consisting of, antibody fragmentsor variants thereof), may be coupled or conjugated to a therapeuticmoiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, atherapeutic agent or a radioactive metal ion, e.g., alpha-emitters suchas, for example, ²¹³Bi, or other radioisotopes such as, for example,¹⁰³Pd, ¹³⁵Xe, ¹³¹I, ⁶⁸Ge, ⁵⁷Co, ⁶⁵ Zn, ⁸⁵Sr, ³²P, ³⁵S, ⁹⁰Y, ¹⁵³Sm,¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, ⁹⁰Y, ¹¹⁷Tin, ¹⁸⁶Re, ¹⁸⁸Re and¹⁶⁶Ho. In specific embodiments, an antibody or fragment thereof isattached to macrocyclic chelators that chelate radiometal ions,including but not limited to, ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and ¹⁵³Sm, topolypeptides. In specific embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). Inother specific embodiments, the DOTA is attached to the an antibody ofthe invention or fragment thereof via a linker molecule. Examples oflinker molecules useful for conjugating DOTA to a polypeptide arecommonly known in the art—see, for example, DeNardo et al., Clin CancerRes. 4(10):2483-90, 1998; Peterson et al., Bioconjug. Chem. 10(4):553-7,1999; and Zimmerman et al., Nucl. Med. Biol. 26(8):943-50, 1999 whichare hereby incorporated by reference in their entirety.

A cytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Examples include, but are not limited to, paclitaxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, thymidine kinase, endonuclease,RNAse, and puromycin and frragments, variants or homologs thereof.Therapeutic agents include, but are not limited to, antimetabolites(e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, andanthramycin (AMC)), and anti-mitotic agents (e.g., vincristine andvinblastine).

Techniques known in the art may be applied to label antibodies of theinvention. Such techniques include, but are not limited to, the use ofbifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065;5,714,711; 5,696,239; 5,652,371; 5,505,931; 5,489,425; 5,435,990;5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contentsof each of which are hereby incorporated by reference in its entirety)and direct coupling reactions (e.g., Bolton-Hunter and Chloramine-Treaction).

The antibodies of the invention which are conjugates can be used formodifying a given biological response, the therapeutic agent or drugmoiety is not to be construed as limited to classical chemicaltherapeutic agents. For example, the drug moiety may be a protein orpolypeptide possessing a desired biological activity. Such proteins mayinclude, but are not limited to, for example, a toxin such as abrin,ricin A, alpha toxin, pseudomonas exotoxin, or diphtheria toxin,saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin andcholera toxin; a protein such as tumor necrosis factor,alpha-interferon, beta-interferon, nerve growth factor, platelet derivedgrowth factor, tissue plasminogen activator, an apoptotic agent, e.g.,TNF-alpha, TNF-beta, AIM I (see, International Publication No. WO97/35899), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574(1994)), VEGI (see, International Publication No. WO 99/23105), athrombotic agent or an anti-angiogenic agent, e.g., angiostatin orendostatin; or, biological response modifiers such as, for example,lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6(IL-6), granulocyte macrophage colony stimulating factor (GM-CSF),granulocyte colony stimulating factor (G-CSF), or other growth factors.

Antibodies of the invention (including antibody fragments or variantsthereof), may also be attached to solid supports, which are particularlyuseful for immunoassays or purification of the target antigen. Suchsolid supports include, but are not limited to, glass, cellulose,polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Techniques for conjugating a therapeutic moiety to antibodies are wellknown, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody of the invention can be conjugated to asecond antibody to form an antibody heteroconjugate as described bySegal in U.S. Pat. No. 4,676,980, which is incorporated herein byreference in its entirety.

An antibody of the invention (including an other molecules comprising,or alternatively consisting of, an antibody fragment or variantthereof), with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Uses of Antibodies of the Invention

Antibodies of the present invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofTL5 polypeptides in biological samples. See, e.g., Harlow et al.,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988) (incorporated by reference herein in its entirety).

Immunophenotyping

The antibodies of the invention may be utilized for immunophenotyping ofcell lines and biological samples. The translation product of the geneof the present invention may be useful as a cell specific marker, ormore specifically as a cellular marker that is differentially expressedat various stages of differentiation and/or maturation of particularcell types, such as T-cells (e.g., activated T-cells). In otherembodiments, the antibodies of the invention may be useful as tumorsand/or cancer cell markers. Monoclonal antibodies directed against aspecific epitope, or combination of epitopes, will allow for thescreening of cellular populations expressing the marker. Varioustechniques can be utilized using monoclonal antibodies to screen forcellular populations expressing the marker(s), and include magneticseparation using antibody-coated magnetic beads, “panning” with antibodyattached to a solid matrix (i.e., plate), and flow cytometry (See, e.g.,U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.minimal residual disease (D) in acute leukemic patients) and “non-self”cells in transplantations to prevent Graft-versus-Host Disease (GVHD).Alternatively, these techniques allow for the screening of hematopoieticstem and progenitor cells capable of undergoing proliferation and/ordifferentiation, as might be found in human umbilical cord blood.

Epitope Mapping

The present invention provides antibodies (including antibody fragmentsor variants thereof), that can be used to identify epitopes of a TL5polypeptide. In particular, the antibodies of the present invention canbe used to identify epitopes of a human TL5 polypeptide (e.g., SEQ IDNOS:2 and/or 4) or a TL5 polypeptide expressed on human cells; a murineTL5 or a TL5 polypeptide expressed on murine cells; a rat TL5polypeptide receptor or a TL5 polypeptide expressed on rat cells; or amonkey TL5 polypeptide or a TL5 polypeptide expressed on monkey cells,using techniques described herein or otherwise known in the art.Fragments which function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135(1985), further described in U.S. Pat. No. 4,711,211.) Identifiedepitopes of antibodies of the present invention may, for example, beused as vaccine candidates, i.e., to immunize an individual to elicitantibodies against the naturally occuring forms of TL5 polypeptides.

Diagnostic Uses of Antibodies

Labeled antibodies of the invention (including molecules comprising, oralternatively consisting of, antibody fragments or variants thereof)which specifically bind to a TL5 polypeptide can be used for diagnosticpurposes to detect, diagnose, prognose, or monitor diseases and/ordisorders. In specific embodiments, labeled antibodies of the invention(including molecules comprising, or alternatively consisting of,antibody fragments or variants thereof) which specifically bind to a TL5polypeptide can be used for diagnostic purposes to detect, diagnose,prognose, or monitor diseases and/or disorders associated with theaberrant expression and/or activity of a TL5 polypeptide.

The invention provides for the detection of expression of a TL5polypeptide comprising: (a) assaying the expression of a TL5 polypeptidein a biological sample from an individual using one or more antibodiesof the invention that specifically binds to a TL5 polypeptide; and (b)comparing the level of a TL5 polypeptide with a standard level of a TL5polypeptide, (e.g., the level in normal biological samples).

The invention provides for the detection of aberrant expression of a TL5polypeptide comprising: (a) assaying the expression of a TL5 polypeptidein a biological sample from an individual using one or more antibodiesof the invention that specifically binds to a TL5 polypeptide; and (b)comparing the level of a TL5 polypeptide with a standard level of a TL5polypeptide, e.g., in normal biological samples, whereby an increase ordecrease in the assayed level of a TL5 polypeptide compared to thestandard level of a TL5 polypeptide is indicative of aberrantexpression.

By “biological sample” is intended any fluids and/or cells obtained froman individual, body fluid, body tissue, body cell, cell line, tissueculture, or other source which may contain a TL5 polypeptide protein ormRNA. Body fluids include, but are not limited to, sera, plasma, urine,synovial fluid, spinal fluid, saliva, and mucous. Tissues samples may betaken from virtually any tissue in the body. Tissue samples may also beobtained from autopsy material. Methods for obtaining tissue biopsiesand body fluids from mammals are well known in the art. Where thebiological sample is to include mRNA, a tissue biopsy is the preferredsource.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a TL5 polypeptide ora TL5 polypeptide receptor (e.g., TR2, TR6, LTβR) in an animal,preferably a mammal and most preferably a human. In one embodiment,diagnosis comprises: a) administering (for example, parenterally,subcutaneously, or intraperitoneally) to a subject an effective amountof a labeled antibody of the invention (including molecules comprising,or alternatively consisting of, antibody fragments or variants thereof)that specifically binds to a TL5 polypeptide; b) waiting for a timeinterval following the administering for permitting the labeled antibodyto preferentially concentrate at sites in the subject where TL5polypeptide is expressed (and for unbound labeled molecule to be clearedto background level); c) determining background level; and d) detectingthe labeled antibody in the subject, such that detection of labeledantibody or fragment thereof above the background level and above orbelow the level observed in a person without the disease or disorderindicates that the subject has a particular disease or disorderassociated with aberrant expression of a TL5 polypeptide or a TL5polypeptide receptor. Background level can be determined by variousmethods including, comparing the amount of labeled molecule detected toa standard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of ⁹⁹Tc. The labeled antibody willthen preferentially accumulate at the location of cells which containthe specific protein. In vivo tumor imaging is described in S. W.Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies andTheir Fragments.” (Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., MassonPublishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In one embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disorder, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patient using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

In specific embodiments, antibodies of the present invention may be usedto diagnose, or monitor the progression of, an autoimmune disorder, animunodeficiency, graft rejection, graft vs. host disease, and/or cancer,particularly those diseases and/or disorders described in the“Therapuetic Uses of Antibodies” sections below.

Therapeutic Uses of Antibodies

One or more antibodies of the present invention (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof) that specifically bind to TL5 may be used locally orsystemically in the body as a therapeutic. The present invention isfurther directed to antibody-based therapies which involve administeringantibodies of the invention (including molecules comprising, oralternatively consisting of, antibody fragments or variants thereof) toan animal, preferably a mammal, and most preferably a human, forpreventing or treating one or more of the disclosed diseases, disorders,or conditions. Therapeutic compounds of the invention include, but arenot limited to, antibodies of the invention and nucleic acids encodingantibodies (and anti-idiotypic antibodies) of the invention as describedherein. In one embodiment, the antibodies of the invention can be usedto treat, ameliorate or prevent diseases, disorders or conditions,including, but not limited to, any one or more of the diseases,disorders, or conditions described herein. The treatment and/orprevention of diseases, disorders, or conditions includes, but is notlimited to, alleviating symptoms associated with those diseases,disorders or conditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

Therapeutic and Diagnostic Uses of Antibodies for Treating AutoimmuneDisorders and Immunodeficiencies

In highly preferred embodiments, antibodies of the invention, e.g.,antibodies of the invention that bind a TL5 polypeptide and inhibitproliferation and/or differentiation of TL5 and/or TL5 receptorexpressing cells, are used to diagnose, treat, prevent or ameliorateautoimmune diseases, disorders, or conditions associated with suchdiseases or disorders. In specific embodiments, antibodies of theinvention are used to inhibit the progression of an autoimmune responseand other related disorders. Autoimmune disorders and related disorders,include, but are not limited to, autoimmune hemolytic anemia, autoimmuneneonatal thrombocytopenia, idiopathic thrombocytopenia purpura,autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome,dermatitis, allergic encephalomyelitis, myocarditis, relapsingpolychondritis, ulcerative colitis, dense deposit disease, rheumaticheart disease, glomerulonephritis (e.g., IgA nephropathy), pemphigusvulgaris, discoid lupus, Multiple Sclerosis, Neuritis, UveitisOphthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenleinpurpura), Reiter's Disease, Stiff-Man Syndrome, Autoimmune PulmonaryInflammation, Guillain-Barre Syndrome, insulin dependent diabetesmellitis, and autoimmune inflammatory eye, autoimmune thyroiditis,hypothyroidism (i.e., Hashimoto's thyroiditis), systemic lupuserhythematosus, Goodpasture's syndrome, Pemphigus, Receptorautoimmunities such as, for example, (a) Graves' Disease, (b) MyastheniaGravis, and (c) insulin resistance, autoimmune hemolytic anemia,autoimmune thrombocytopenic purpura , rheumatoid arthritis,schleroderrna with anti-collagen antibodies, mixed connective tissuedisease, polymyositis/dermatomyositis, pernicious anemia, idiopathicAddison's disease, infertility, glomerulonephritis such as primaryglomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjogren'ssyndrome, diabetes millitus, and adrenergic drug resistance (includingadrenergic drug resistance with asthma or cystic fibrosis), chronicactive hepatitis, primary biliary cirrhosis, other endocrine glandfailure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria,atopic dermatitis, asthma, inflammatory myopathies, graft v. hostdiseases (GVHD) and other inflammatory, granulamatous, degenerative, andatrophic disorders).

In a specific embodiment, antibodies of the invention are be used todiagnose, treat, inhibit, prognose, diagnose or prevent rheumatoidarthritis.

In another specific embodiment, antibodies of the invention are used todiagnose, treat, inhibit, prognose, diagnose or prevent systemic lupuserythematosis.

In another specific embodiment, antibodies of the invention are used todiagnose, treat, inhibit, prognose, diagnose or prevent graft v. hostdiseases (GVHD).

Additionally, the antibodies of the invention can be used to diagnose,treat, inhibit or prevent diseases, disorders or conditions associatedwith immunodeficiencies including, but not limited to, severe combinedimmunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminasedeficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton'sdisease, congenital agammaglobulinemia, X-linked infantileagammaglobulinemia, acquired agammaglobulinemia, adult onsetagammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia,hypogammaglobulinemia, transient hypogammaglobulinemia of infancy,unspecified hypogammaglobulinemia, agammaglobulinemia, common variableimmunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS),X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiencywith hyper IgM, selective IgA deficiency, IgG subclass deficiency (withor without IgA deficiency), antibody deficiency with normal or elevatedIgs, immunodeficiency with thymoma, Ig heavy chain deletions, kappachain deficiency, B cell lymphoproliferative disorder (BLPD), selectiveIgM immunodeficiency, recessive agammaglobulinemia (Swiss type),reticular dysgenesis, neonatal neutropenia, autoimmune neutropenia,severe congenital leukopenia, thymic alymphoplasia-aplasia or dysplasiawith immunodeficiency, ataxia-telangiectasia, short limbed dwarfism,X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome-combinedimmunodeficiency with Igs, purine nucleoside phosphorylase deficiency(PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severecombined immunodeficiency.

In another specific embodiment, antibodies of the invention are used todiagnose, treat, or prevent graft rejection and inflammation and for thetreatment of arthritis.

Therapeutic Uses of Antibodies for Treating Cancers

In highly preferred embodiments, antibodies of the invention, e.g.,antibodies of the invention that bind a TL5 polypeptide and inhibitproliferation of TL5 and/or TL5 receptor expressing cells are used todiagnose, treat, prevent or ameliorate cancer. In specific embodiments,antibodies of the invention are used to inhibit the progression ormetastasis of cancers and other related disorders. Cancers and relateddisorders, include, but are not limited to, colon cancer, cervicalcancer, leukemia (including acute leukemias (e.g., acute lymphocyticleukemia, acute myelocytic leukemia (including myeloblastic,promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) andchronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia andchronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.,Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumorsincluding, but not limited to, sarcomas and carcinomas such asfibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, testicular tumor, lungcarcinoma, small cell lung carcinoma, bladder carcinoma, epithelialcarcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

In specific embodiments, antibodies of the invention used to treat theaforementioned cancers are administered with or conjugated to acytotoxic and/or chemotherapeutic agent.

Additional Therapeutic Uses of Antibodies

In another embodiment, the invention provides methods and compositionsfor inhibiting the growth of or killing TL5 expressing cells,comprising, or alternatively consisting of, administering to an animalin which such inhibition of growth or killing of TL5 expressing cells isdesired, antibody or antibody compositions of the invention (e.g.,antibody fragments and variants, antibody mixtures, antibody multimers,fusion proteins of the invention, and antibodies in combination with orconjugated to other therapeutic compounds such as chemotherapeuticagents) in an amount effective to inhibit the growth of or kill TL5expressing cells.

In one aspect, the present invention is directed to a method forenhancing apoptosis induced by TL5, which involves administering to acell which expresses a TL5 receptor polypeptide an effective amount ofan antibody of the invention, capable of inducing or increasing TL5mediated signaling. Preferably, TL5 mediated signaling is increased orinduced by an antibody of the invention to treat a disease whereindecreased apoptosis or decreased cytokine and adhesion moleculeexpression is exhibited.

In a further aspect, the present invention is directed to a method forinhibiting apoptosis induced by TL5, which involves administering to acell which expresses a TL5 receptor polypeptide, an effective amount ofan antibody of the invention, capable of decreasing TL5 mediatedsignaling. Preferably, TL5 mediated signaling is decreased to treat adisease wherein increased apoptosis is exhibited.

The antibodies of the invention can be used to diagnose, treat,ameliorate or prevent diseases, disorders or conditions associated withaberrant expression and/or activity of TL5 and/or a TL5 receptor,including, but not limited to, any one or more of the diseases,disorders, or conditions described herein. The treatment and/orprevention of diseases, disorders, or conditions associated withaberrant TL5 expression and/or activity or TL5 receptor expressionand/or activity includes, but is not limited to, alleviating symptomsassociated with those diseases, disorders or conditions. Antibodies ofthe invention may be provided in pharmaceutically acceptablecompositions as known in the art or as described herein.

Further, antibodies of the present invention (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof) which inhibit TL5-mediated biological activities(e.g., the induction of apoptosis in TL5 receptor expressing cells; orthe ability to deliver costimulatory signals to T cells; or to induce Tcells to proliferate or differentiate (e.g., the ability to induce naiveCD45RA+ cells to secrete type 1 cytokines such as interferon gamma)) canbe administered to an animal to diagnose, treat, prevent or ameliorate adisease or disorder associated aberrant TL5 expression or functionand/or aberrant TL5 receptor expression or function. These antibodiesmay inhibit or reduce either all or a subset of the biologicalactivities of TL5, for example, by inducing a conformational change inTL5. In a specific embodiment, an antibody of the present invention thatinhibits TL5 activity by at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 99%, or 100% relative to TL5 activity in absence ofthe antibody is administered to an animal to diagnose, treat, prevent orameliorate a disease or disorder associated with aberrant TL5 expressionor function and/or aberrant TL5 receptor expression or function. Inanother embodiment, a combination of antibodies, a combination ofantibody fragments, a combination of antibody variants, or a combinationof antibodies, antibody fragments and/or antibody variants that inhibitTL5 activity by at least 5%, at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 99%, or 100% relative to TL5 activity in absence of the saidantibodies or antibody fragments and/or antibody variants isadministered to an animal to diagnose, treat, prevent or ameliorate adisease or disorder associated with aberrant TL5 expression or functionand/or aberrant TL5 receptor expression or function.

In a specific embodiment, an antibody of the present invention(including molecules comprising, or alternatively consisting of,antibody fragments or variants thereof) that inhibit or downregulates,in full or in part, TL5 activity (e.g., stimulation of apoptosis) by atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 99%, or100% relative to TL5 activity in absence of the antibody is administeredto an animal to diagnose, treat, prevent or ameliorate a disease ordisorder described herein, particularly an autoimmune disease (such assystemic lupus erythematosus and rheumatoid arthritis), and even moreparticularly an autoimmune disease that presents a type I T cell profile(such as myasthenia gravis, multiple sclerosis, and autoimmunediabetes), graft versus host disease and transplant rejection. Inanother embodiment, a combination of antibodies, a combination ofantibody fragments, a combination of antibody variants, or a combinationof antibodies, antibody fragments, and/or variants that inhibit ordownregulate TL5 activity by at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 99%, or 100% relative to TL5 activity in absence ofsaid antibodies, antibody fragments, and/or antibody variants areadministered to an animal to diagnose, treat, prevent or ameliorate adisease or disorder described herein, particularly an autoimmune disease(such as systemic lupus erythematosus and rheumatoid arthritis), andeven more particularly an autoimmune disease that presents a type I Tcell profile (such as myasthenia gravis, multiple sclerosis, andautoimmune diabetes), graft versus host disease and transplantrejection.

Further, antibodies of the present invention (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof) which activate TL5-mediated biological activities(e.g., the induction of apoptosis in TL5 receptor expressing cells; orthe ability to deliver costimulatory signals to T cells; or to induce Tcells to proliferate or differentiate (e.g., the ability to induce naïveCD45RA+ cells to secrete type 1 cytokines such as interferon gamma)) canbe administered to an animal to diagnose, treat, prevent or ameliorate adisease or disorder described herein, particularly cancers and otherhyperproliferative disorders, especially cancers which express TL5and/or a TL5 receptor. These antibodies may potentiate or activateeither all or a subset of the biological activities of TL5, for example,by inducing a conformational change in TL5. In a specific embodiment, anantibody of the present invention that increases TL5 activity by atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 99%, atleast two-fold, at least three-fold, at least four fold, at least fivefold, at least ten-fold, at least twenty-fold, at least fifty-fold, orat least one hundred-fold relative to TL5 activity in absence of theantibody is administered to an animal to diagnose, treat, prevent orameliorate a disease or disorder. In another embodiment, a combinationof antibodies, a combination of antibody fragments, a combination ofantibody variants, or a combination of antibodies, antibody fragmentsand/or antibody variants that increase TL5 activity by at least 5%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 99%, at least two-fold,at least three-fold, at least four fold, at least five fold, at leastten-fold, at least twenty-fold, at least fifty-fold, or at least onehundred-fold relative to TL5 activity in absence of the said antibodiesor antibody fragments and/or antibody variants is administered to ananimal to diagnose, treat, prevent or ameliorate a disease or disorder.

Further, antibodies of the present invention (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof) which activate TL5-mediated biological activities(e.g., the induction of apoptosis in TL5 receptor expressing cells; orthe ability to deliver costimulatory signals to T cells; or to induce Tcells to proliferate or differentiate (e.g., the ability to induce naiveCD45RA+ cells to secrete type 1 cytokines such as interferon gamma)) canbe administered to an animal to diagnose, treat, prevent or ameliorate adisease or disorder associated with aberrant TL5 expression, function,or aberrant TL5 receptor expression or function. These antibodies maypotentiate or activate either all or a subset of the biologicalactivities of TL5, for example, by inducing a conformational change inTL5. In a specific embodiment, an antibody of the present invention thatincreases TL5 activity by at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 99%, at least two-fold, at least three-fold, atleast four fold, at least five fold, at least ten-fold, at leasttwenty-fold, at least fifty-fold, or at least one hundred-fold relativeto TL5 activity in absence of the antibody is administered to an animalto diagnose, treat, prevent or ameliorate a disease or disorderassociated with aberrant TL5 expression, function, or aberrant TL5receptor expression or function. In another embodiment, a combination ofantibodies, a combination of antibody fragments, a combination ofantibody variants, or a combination of antibodies, antibody fragmentsand/or antibody variants that increase TL5 activity by at least 5%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 99%, at least two-fold,at least three-fold, at least four fold, at least five fold, at leastten-fold, at least twenty-fold, at least fifty-fold, or at least onehundred-fold relative to TL5 activity in absence of the said antibodiesor antibody fragments and/or antibody variants is administered to ananimal to diagnose, treat, prevent or ameliorate a disease or disorderassociated with aberrant TL5 expression, function, or aberrant TL5receptor expression or function.

Antibodies of the present invention (including molecules comprising, oralternatively consisting of, antibody fragments or variants thereof)that function as agonists or antagonists of a TL5, preferably of TL5signal transduction, can be administered to an animal to diagnose,treat, prevent or ameliorate a disease or disorder associated aberrantTL5 expression, function, or aberrant TL5 receptor expression orfunction. For example, antibodies of the invention which mimic theaction of TL5 binding to the TL5 receptor, in full or in part, (e.g.antibodies that act as TL5 agonists), may be administered to an animalto diagnose, treat, prevent or ameliorate a disease or disorderassociated aberrant TL5 expression, function, or aberrant TL5 receptorexpression or function. As an alternative example, antibodies of theinvention which disrupt or prevent the interaction between TL5 and itsreceptor or inhibit, reduce, or prevent signal transduction through oneor more TL5s, may be administered to an animal to diagnose, treat,prevent or ameliorate a disease or disorder associated with aberrant TL5expression, function, or aberrant TL5 receptor expression or function.Antibodies of the invention which do not prevent TL5 from binding itsligand but inhibit or downregulate TL5 signal transduction can beadministered to an animal to diagnose, treat, prevent or ameliorate adisease or disorder associated with aberrant TL5 expression, lack of TL5function, aberrant TL5 receptor expression, or lack of TL5 receptorfunction. The ability of an antibody of the invention to enhance,inhibit, upregulate or downregulate TL5 signal transduction may bedetermined by techniques described herein or otherwise known in the art.For example, TL5-induced receptor activation and the activation ofsignaling molecules can be determined by detecting the association ofadaptor proteins with the TL5 receptors, by immunoprecipitation followedby western blot analysis (for example, as described herein).

In one embodiment, therapeutic or pharmaceutical compositions of theinvention are administered to an animal to diagnose, treat, prevent orameliorate a disease or disorder diseases associated with increasedapoptosis including, but not limited to, AIDS, neurodegenerativedisorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophiclateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration),myelodysplastic syndromes (such as aplastic anemia), ischemic injury(such as that caused by myocardial infarction, stroke and reperfusioninjury), toxin-induced liver disease (such as that caused by alcohol),septic shock, cachexia and anorexia. In another embodiment, therapeuticor pharmaceutical compositions of the invention are administered to ananimal to diagnose, treat, prevent or ameliorate bone marrow failure,for example, aplastic anemia and myelodysplastic syndrome.

Therapeutic or pharmaceutical compositions of the invention, may also beadministered to diagnose, treat, prevent, or ameliorate organ rejectionor graft-versus-host disease (GVHD) and/or conditions associatedtherewith. Organ rejection occurs by host immune cell destruction of thetransplanted tissue through an immune response. Similarly, an immuneresponse is also involved in GVHD, but, in this case, the foreigntransplanted immune cells destroy the host tissues. Cellular deathinduced by immune cell effector functions is apoptotic death. Thus, theadministration of antibodies of the invention, (e.g., those that inhibitapoptosis or those that antagonize the costimulatory activity of TL5 onT cells), may be an effective therapy in preventing organ rejection orGVHD.

In another embodiment, therapeutic or pharmaceutical compositions of theinvention are administered to an animal to diagnose, treat, prevent orameliorate infectious diseases. Infectious diseases include diseasesassociated with yeast, fungal, viral and bacterial infections. Virusesassociated with viral infections which can be treated or prevented inaccordance with this invention include, but are not limited to,retroviruses (e.g., human T-cell lymphotrophic virus (HTLV) types I andII and human immunodeficiency virus (HIV)), herpes viruses (e.g., herpessimplex virus (HSV) types I and II, Epstein-Barr virus, HHV6-HHV8, andcytomegalovirus), arenavirues (e.g., lassa fever virus), paramyxoviruses(e.g., morbillivirus virus, human respiratory syncytial virus, mumps,and pneumovirus), adenoviruses, bunyaviruses (e.g., hantavirus),cornaviruses, filoviruses (e.g., Ebola virus), flaviviruses (e.g.,hepatitis C virus (HCV), yellow fever virus, and Japanese encephalitisvirus), hepadnaviruses (e.g., hepatitis B viruses (HBV)),orthomyoviruses (e.g., influenza viruses A, B and C), papovaviruses(e.g., papillomavirues), picornaviruses (e.g., rhinoviruses,enteroviruses and hepatitis A viruses), poxviruses, reoviruses (e.g.,rotavirues), togaviruses (e.g., rubella virus), rhabdoviruses (e.g.,rabies virus). Microbial pathogens associated with bacterial infectionsinclude, but are not limited to, Streptococcus pyogenes, Streptococcuspneumoniae, Neisseria gonorrhoea, Neisseria meningitidis,Corynebacterium diphtheriae, Clostridium botulinum, Clostridiumperfringens, Clostridium tetani, Haemophilus influenzae, Klebsiellapneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromotis,Staphylococcus aureus, Vibrio cholerae, Escherichia coli, Pseudomonasaeruginosa, Campylobacter (Vibrio) fetus, Campylobacter jejuni,Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersiniaenterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigelladysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium,Treponema pallidum, Treponema pertenue, Treponema carateneum, Borreliavincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae,Mycobacterium tuberculosis, Toxoplasma gondii, Pneumocystis carinii,Francisella tularensis, Brucella abortus, Brucella suis, Brucellamelitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsiatsutsugumushi, Chlamydia spp., and Helicobacter pylori.

In another embodiment, antibodies and antibody compositions of thepresent invention are used to diagnose, treat, prevent, or amelioratediseases associated with increased apoptosis including, but not limitedto, AIDS, neurodegenerative disorders (such as, Amyotrophic lateralsclerosis, Retinitis pigmentosa, Cerebellar degeneration), brain tumoror prion associated disease); autoimmune disorders (such as, multiplesclerosis, Rheumatoid Arthritis, Sjogren's syndrome, Hashimoto'sthyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes(such as aplastic anemia), graft v. host disease, ischemic injury (suchas that caused by myocardial infarction, stroke and reperfusion injury),liver injury (e.g., hepatitis related liver injury, ischemia/reperfusioninjury, cholestosis (bile duct injury) and liver cancer); toxin-inducedliver disease (such as that caused by alcohol), septic shock, cachexiaand anorexia. In preferred embodiments, TL5 antagonistic antibodies,(e.g., antibodies that bind one or more TL5 polypeptides and prevent TL5from binding to the TL5 receptors) are used to treat the diseases anddisorders listed above.

Many of the pathologies associated with HIV are mediated by apoptosis,including HIV-induced nephropathy and HIV encephalitis. Thus, inadditional preferred embodiments, antibodies, preferably antagonisticTL5 antibodies, of the invention are used to treat AIDS and pathologiesassociated with AIDS. Another embodiment of the present invention isdirected to the use of antibodies of the invention to reduceTL5-mediated death of T cells in HIV-infected patients.

In additional embodiments, antibodies of the present invention,particularly antagonistic anti-TL5 antibodies, are administered incombination with other inhibitors of T cell apoptosis. For example,Fas-mediated apoptosis has been implicated in loss of T cells in HIVindividuals (Katsikis et al., J. Exp. Med. 181:2029-2036, 1995). Thus, apatient susceptible to both Fas ligand mediated and TL5 mediated T celldeath may be treated with both an agent that blocks TL5/TL5 receptorinteractions and an agent that blocks Fas-ligand/Fas interactions.Suitable agents for blocking binding of Fas-ligand to Fas include, butare not limited to, soluble Fas polypeptides; mulitmeric forms ofsoluble Fas polypeptides (e.g., dimers of sFas/Fc); anti-Fas antibodiesthat bind Fas without transducing the biological signal that results inapoptosis; anti-Fas-ligand antibodies that block binding of Fas-ligandto Fas; and muteins of Fas-ligand that bind Fas but do not transduce thebiological signal that results in apoptosis. Preferably, the antibodiesemployed according to this method are monoclonal antibodies. Examples ofsuitable agents for blocking Fas-ligand/Fas interactions, includingblocking anti-Fas monoclonal antibodies, are described in Internationalapplication publication number WO 95/10540, hereby incorporated byreference.

Suitable agents, which also block binding of TL5 to a TL5 receptor(e.g., TR6 (described in WO98/30694, WO2000/52028, WO2002/18622, and SEQID NO:49), LTβR (See, e.g., Genbank™ Accesion Numbers L04270 and P36941and SEQ ID NO:47), and TR2 (described in WO96/34095, WO98/18824,WO00/56405 and SEQ ID NO:48)) that may be administered with theantibodies of the present invention include, but are not limited to,soluble TL5 receptor polypeptides; multimeric forms of soluble TL5receptor polypeptides; and TL5 antibodies that bind the TL5 withouttransducing the biological signal that results in apoptosis, anti-TL5antibodies that block binding of TL5 to one or more TL5 receptors, andmuteins of TL5 that bind TL5 receptors but do not transduce thebiological signal that results in apoptosis.

Antibodies and antibody compositions of the invention may be useful fortreating inflammatory diseases, such as rheumatoid arthritis,osteoarthritis, psoriasis, septicemia, and inflammatory bowel disease.

Antibodies and antibody compositions of the invention are useful in thediagnosis and treatment or prevention of a wide range of diseases and/orconditions. Such diseases and conditions include, but are not limitedto, cancer (e.g., immune cell related cancers, breast cancer, prostatecancer, ovarian cancer, follicular lymphoma, cancer associated withmutation or alteration of p53, brain tumor, bladder cancer,uterocervical cancer, colon cancer, colorectal cancer, non-small cellcarcinoma of the lung, small cell carcinoma of the lung, stomach cancer,etc.), lymphoproliferative disorders (e.g., lymphadenopathy), microbial(e.g., viral, bacterial, etc.) infection (e.g., HIV-1 infection, HIV-2infection, herpesvirus infection (including, but not limited to, HSV-1,HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenovirus infection, poxvirusinfection, human papilloma virus infection, hepatitis infection (e.g.,HAV, HBV, HCV, etc.), Helicobacter pylori infection, invasiveStaphylococcia, etc.), parasitic infection, nephritis, bone disease(e.g., osteoporosis), atherosclerosis, pain, cardiovascular disorders(e.g., neovascularization, hypovascularization or reduced circulation(e.g., ischemic disease (e.g., myocardial infarction, stroke, etc.))),AIDS, allergy, inflammation, neurodegenerative disease (e.g.,Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,pigmentary retinitis, cerebellar degeneration, etc.), graft rejection(acute and chronic), graft vs. host disease, diseases due toosteomyelodysplasia (e.g., aplastic anemia, etc.), joint tissuedestruction in rheumatism, liver disease (e.g., acute and chronichepatitis, liver injury, and cirrhosis), autoimmune disease (e.g.,multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus,autoimmune lymphoproliferative syndrome (ALPS), immune complexglomerulonephritis, autoimmune diabetes, autoimmune thrombocytopenicpurpura, Grave's disease, Hashimoto's thyroiditis, etc.), cardiomyopathy(e.g., dilated cardiomyopathy), diabetes, diabetic complications (e.g.,diabetic nephropathy, diabetic neuropathy, diabetic retinopathy),influenza, asthma, psoriasis, glomerulonephritis, septic shock, andulcerative colitis.

Antibodies and antibody compositions of the invention are useful inpromoting angiogenesis, wound healing (e.g., wounds, burns, and bonefractures).

Antibodies and antibody compositions of the invention are also useful asan adjuvant to enhance immune responsiveness to specific antigen, suchas in anti-viral immune responses.

More generally, antibodies and antibody compositions of the inventionare useful in regulating (i.e., elevating or reducing) immune response.For example, antibodies and antibody compositions of the invention maybe useful in preparation or recovery from surgery, trauma, radiationtherapy, chemotherapy, and transplantation, or may be used to boostimmune response and/or recovery in the elderly and immunocompromisedindividuals. Alternatively, antibodies and antibody compositions of theinvention are useful as immunosuppressive agents, for example in thetreatment or prevention of autoimmune disorders. In specificembodiments, antibodies and antibody compositions of the invention areused to treat or prevent chronic inflammatory, allergic or autoimmuneconditions, such as those described herein or are otherwise known in theart.

Therapeutic/Prophylactic Compositions and Administration

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of antibody (orfragment or variant thereof) or pharmaceutical composition of theinvention, preferably an antibody of the invention. In a preferredaspect, an antibody or fragment or variant thereof is substantiallypurified (i.e., substantially free from substances that limit its effector produce undesired side-effects). The subject is preferably an animal,including but not limited to, animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably a human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administerantibody or fragment or variant thereof of the invention, e.g.,encapsulation in liposomes, microparticles, microcapsules, recombinantcells capable of expressing the antibody or antibody fragment,receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem.262:4429-4432 (1987)), construction of a nucleic acid as part of aretroviral or other vector, etc. Methods of introduction include, butare not limited to, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, epidural, and oral routes. Thecompositions may be administered by any convenient route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local. In addition, it may bedesirable to introduce the pharmaceutical compositions of the inventioninto the central nervous system by any suitable route, includingintraventricular and intrathecal injection; intraventricular injectionmay be facilitated by an intraventricular catheter, for example,attached to a reservoir, such as an Ommaya reservoir. Pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. Preferably,when administering a protein, including an antibody, of the invention,care must be taken to use materials to which the protein does notabsorb.

In another embodiment, the composition can be delivered in a vesicle, inparticular a liposome (see Langer, Science 249:1527-1535 (1990); Treatet al., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 3 17-327; see generally ibid.).

In yet another embodiment, the composition can be delivered in acontrolled release system. In one embodiment, a pump may be used (seeLanger, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:20 1 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem.23:71 (1983); see also Levy et al., Science 228:190 (1985); During etal., Ann. Neurol. 25:35 1 (1989); Howard et al., J. Neurosurg. 7 1:105(1989)). In yet another embodiment, a controlled release system can beplaced in proximity of the therapeutic target, i.e., the brain, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1535 (1990)).

In a specific embodiment where the composition of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of an antibodyor a fragment thereof, and a pharmaceutically acceptable carrier. In aspecific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will contain a therapeutically effective amount of theantibody or fragment thereof, preferably in purified form, together witha suitable amount of carrier so as to provide the form for properadministration to the patient. The formulation should suit the mode ofadministration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocamne to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compositions of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the composition of the invention which will be effectivein the treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a polypeptide ofthe invention can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of therapeutic orpharmaceutical compositions of the invention may be reduced by enhancinguptake and tissue penetration (e.g., into the brain) of the antibodiesby modifications such as, for example, lipidation.

Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments, or variants, (e.g., derivatives), or nucleicacids, are administered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies of the invention (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof) that specifically bind to one or more TL5polypeptides, or polynucleotides encoding antibodies that specificallybind to one or more TL5 polypeptides, for both immunoassays and therapyof disorders related to TL5 polynucleotides or polypeptides, includingfragments thereof. Such antibodies will preferably have an affinity forTL5 polypeptides and/or TL5 polypeptide fragments. Preferred bindingaffinities include those with a dissociation constant or K_(D) of lessthan or equal to 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M,5×10⁻⁵ M, or 10⁻⁵ M. More preferably, antibodies of the invention bindTL5 polypeptides or fragments or variants thereof with a dissociationconstant or K_(D) less than or equal to 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷M, 5×10⁻⁸ M, or 10⁻⁸ M. Even more preferably, antibodies of theinvention bind TL5 polypeptides or fragments or variants thereof with adissociation constant or K_(D) less than or equal to 5×10⁻⁹ M, 10⁻⁹ M,5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×⁻¹³ M,10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M. In a preferredembodiment, antibodies of the invention inhibit proliferation,differentiation, and/or apoptosis of TL5 receptor expressing cells. Inan additional preferred embodiment, antibodies of the invention inducedifferentiation of TL5 receptor expressing cells.

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalent and non-covalent conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionuclides, or toxins. See, e.g.,PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387.

The antibody and antibody compositions of the invention may beadministered alone or in combination with other therapeutic agents,including but not limited to chemotherapeutic agents, antibiotics,antivirals, anti-retroviral agents, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents andcytokines. Combinations may be administered either concomitantly, e.g.,as an admixture, separately but simultaneously or concurrently; orsequentially. This includes presentations in which the combined agentsare administered together as a therapeutic mixture, and also proceduresin which the combined agents are administered separately butsimultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

Combination Therapies with Anti-TL5 Antibodies, Immunomodulatory Agents,Antiviral Drugs, and/or Chemotherapeutic Agents

Anti-TL5 antibodies may be administered in combination with otheranti-TL5 antibodies, TL5, and/or chemotherapeutics.

In specific embodiments, an antibody of the invention that specificallybinds TL5 is used or administered in combination with a second antibodythat specifically binds TL5. In another embodiment, the antibodiesspecific for TL5 are agonistic antibodies that stimulate apoptosis,differentiation and/or activation of TL5 expressing cells (e.g. Tcells). In a specific embodiment, the combination of anti-TL5 treatmentsitmulates more apoptosis, differentiation and/or activation of TL5expressing cells than either anti-TL5 antibody treatment alone. Theanti-TL5 antibodies can be administered either simultaneously,sequentially, or a combination of simultaneous or sequentialadministration throughout the dosage regimen. In another specificembodiment anti-TL5 antibodies are used or administered in combinationwith a chemotherapeutic drug, antiviral drug, and/or immunomodulatorydrug. In a particular embodiment, the synergistic inhibition ofproliferation, differentiation, and/or apoptosis or stimulation ofdifferentiation resulting from anti-TL5 antibody treatment, is moreevident or more pronounced when the anti-TL5 antibodies are used oradministered in combination with a chemotherapeutic agent, antiviraldrug, immunomodulatory drug, and/or a cross-linking reagent.

In another embodiment, the second antibody specific for TL5 is anantagonistic antibody that inhibits proliferation, differentiation,and/or apoptosis of TL5 expressing cells. In a specific embodiment, thecombination of anti-TL5 treatment inhibits more proliferation,differentiation, and/or apoptosis of TL5 expressing cells than eitheranti-TL5 antibody treatment alone.

In additional embodiments, anti-TL5 antibodies of the present inventionmay be administered in combination with a soluble form of otherTNF-family receptors which include, but are not limited to, TR2, LTα,TR6, and LTβR.

In other embodiments, antibody compositions of the invention may beadministered in combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe albumin fusion proteins and/or polynucleotides of the invention,include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE™,DAPSONE™, PENTAMIDINE™, ATOVAQUONE™, ISONIAZID™, RIFAMPIN™,PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™, CLARITHROMYCIN™, AZITHROMYCIN™,GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™, FLUCONAZOLE™, ITRACONAZOLE™,KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™, PYRIMETHAMINE™, LEUCOVORIN™,NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™ (sargramostim/GM-CSF).

In a preferred embodiment, compositions of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents that may be administered with the compositionsof the invention include, but are not limited to, antibiotic derivatives(e.g., doxorubicin (adriamycin), bleomycin, daunorubicin, anddactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g.,fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2b,glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxicagents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside,cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin,busulfan, cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, etoposide,Topotecan, 5-Fluorouracil, paclitaxel (Taxol™), Cisplatin, Cytarabine,and IFN-gamma, irinotecan (Camptosar, CPT-11), and gemcitabine(GEMZAR™)).

In a specific embodiment, antibody and antibody compositions of theinvention are administered in combination with steroids, cyclosporine,cyclosporine analogs, cyclophosphamide methylprednisone, prednisone,azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressiveagents that act by suppressing the function of responding T cells. Otherimmunosuppressive agents that may be administered in combination withthe compositions of the invention include, but are not limited to,prednisolone, methotrexate, thalidomide, methoxsalen, rapamycin,leflunomide, mizoribine (BREDININ™), brequinar, deoxyspergualin, andazaspirane (SKF 105685), ORTHOCLONE OKT® 3 (muromonab-CD3), SANDIMMUNE™,NEORAL™, SANGDYA™ (cyclosporine), PROGRAF® (FK506, tacrolimus),CELLCEPT® (mycophenolate motefil, of which the active metabolite ismycophenolic acid), IMURAN™ (azathioprine), glucocorticosteroids,adrenocortical steroids such as DELTASONE™ (prednisone) and HYDELTRASOL™(prednisolone), FOLEX™ and MEXATE™ (methotrxate), OXSORALEN-ULTRA™(methoxsalen) and RAPAMUNE™ (sirolimus). In a specific embodiment,immunosuppressants may be used to prevent rejection of organ or bonemarrow transplantation.

In other embodiments, the compositions of the invention are administeredin combination with immunostimulants including, but not limited to,levamisole (e.g., ERGAMISOL™), isoprinosine (e.g. INOSIPLEX™),interferons (e.g. interferon alpha), and interleukins (e.g., IL-2).

In one embodiment, the compositions of the invention are administered incombination with other members of the TNF family or antibodies specificfor TNF receptor family members. TNF, TNF-related or TNF-like moleculesthat may be administered with the compositions of the invention include,but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha(LT-alpha, also known as TNF-beta), LT-beta (found in complexheterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL,DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328),APRIL (J. Exp. Med. 188(6):1185-1190), endokine-alpha (InternationalPublication No. WO 98/07880), TR6 (International Publication No. WO98/30694), OPG, and neutrokine-alpha (International Publication No. WO98/18921, OX40, and nerve growth factor (NGF), and soluble forms of Fas,CD30, CD27, CD40 and 4-IBB, TL5 (International Publication No. WO96/34095), DR3 (International Publication No. WO 97/35904), TRANK, TR9(International Publication No. WO 98/56892),TR10 (InternationalPublication No. WO 98/54202),312C2 (International Publication No. WO98/06842), and TR12, and soluble forms CD154, CD70, and CD153.

Additional Combination Therapies

The antibodies of the invention (including molecules comprising, oralternatively consisting of, antibody fragments or variants thereof) maybe administered alone or in combination with other therapeutic orprophylactic regimens (e.g., radiation therapy, chemotherapy, hormonaltherapy, immunotherapy, anti-tumor agents, anti-angiogenesis andanti-inflammatory agents). Such combinatorial therapy may beadministered sequentially and/or concomitantly.

The invention also encompasses combining the polynucleotides and/orpolypeptides of the invention (and/or agonists or antagonists thereof)with other proposed or conventional hematopoietic therapies. Thus, forexample, the polynucleotides and/or polypeptides of the invention(and/or agonists or antagonists thereof) can be combined with compoundsthat singly exhibit erythropoietic stimulatory effects, such aserythropoietin, testosterone, progenitor cell stimulators, insulin-likegrowth factor, prostaglandins, serotonin, cyclic AMP, prolactin, andtriiodothyzonine. Also encompassed are combinations of the antibody andantibody compositions of the invention with compounds generally used totreat aplastic anemia, such as, for example, methenolene, stanozolol,and nandrolone; to treat iron-deficiency anemia, such as, for example,iron preparations; to treat malignant anemia, such as, for example,vitamin B₁₂ and/or folic acid; and to treat hemolytic anemia, such as,for example, adrenocortical steroids, e.g., corticoids. See e.g.,Resegotti et al., Panminerva Medica, 23:243-248 (1981); Kurtz, FEBSLetters, 14a:105-108 (1982); McGonigle et al., Kidney Int., 25:437-444(1984); and Pavlovic-Kantera, Expt. Hematol., 8(supp. 8) 283-291 (1980),the contents of each of which are hereby incorporated by reference intheir entireties.

Compounds that enhance the effects of or synergize with erythropoietinare also useful as adjuvants herein, and include but are not limited to,adrenergic agonists, thyroid hormones, androgens, hepatic erythropoieticfactors, erythrotropins, and erythrogenins, See for e.g., Dunn, “CurrentConcepts in Erythropoiesis”, John Wiley and Sons (Chichester, England,1983); Kalmani, Kidney Int., 22:383-391 (1982); Shahidi, New Eng. J.Med., 289:72-80 (1973); Urabe et al., J. Exp. Med., 149:1314-1325(1979); Billat et al., Expt. Hematol., 10:135-140 (1982); Naughton etal., Acta Haemat, 69:171-179 (1983); Cognote et al. in abstract 364,Proceedings 7th Intl. Cong. of Endocrinology (Quebec City, Quebec, Jul.1-7, 1984); and Rothman et al., 1982, J. Surg. Oncol., 20:105-108(1982). Methods for stimulating hematopoiesis comprise administering ahematopoietically effective amount (i.e., an amount which effects theformation of blood cells) of a pharmaceutical composition containingpolynucleotides and/or poylpeptides of the invention (and/or agonists orantagonists thereof) to a patient. The polynucleotides and/orpolypeptides of the invention and/or agonists or antagonists thereof isadministered to the patient by any suitable technique, including but notlimited to, parenteral, sublingual, topical, intrapulmonary andintranasal, and those techniques further discussed herein. Thepharmaceutical composition optionally contains one or more members ofthe group consisting of erythropoietin, testosterone, progenitor cellstimulators, insulin-like growth factor, prostaglandins, serotonin,cyclic AMP, prolactin, triiodothyzonine, methenolene, stanozolol, andnandrolone, iron preparations, vitamin B₁₂, folic acid and/oradrenocortical steroids.

In an additional embodiment, the antibody and antibody compositions ofthe invention are administered in combination with hematopoietic growthfactors. Hematopoietic growth factors that may be administered with theantibody and antibody compositions of the invention include, but are notlimited to, LEUKINE™ (SARGRAMOSTIM™) and NEUPOGEN™ (FILGRASTIM™).

In an additional embodiment, the antibody and antibody compositions ofthe invention are administered alone or in combination with ananti-angiogenic agent(s). Anti-angiogenic agents that may beadministered with the antibody and antibody compositions of theinvention include, but are not limited to, Angiostatin (Entremed,Rockville, Md.), Troponin-1 (Boston Life Sciences, Boston, Mass.),anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel(Taxol™), Suramin, Tissue Inhibitor of Metalloproteinase-1, TissueInhibitor of Metalloproteinase-2, VEGI, Plasminogen ActivatorInhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of thelighter “d group” transition metals.

Lighter “d group” transition metals include, for example, vanadium,molybdenum, tungsten, titanium, niobium, and tantalum species. Suchtransition metal species may form transition metal complexes. Suitablecomplexes of the above-mentioned transition metal species include oxotransition metal complexes.

Representative examples of vanadium complexes include oxo vanadiumcomplexes such as vanadate and vanadyl complexes. Suitable vanadatecomplexes include metavanadate and orthovanadate complexes such as, forexample, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten (IV) oxide andtungsten (VI) oxide. Suitable oxo molybdenum complexes includemolybdate, molybdenum oxide, and molybdenyl complexes. Suitablemolybdate complexes include ammonium molybdate and its hydrates, sodiummolybdate and its hydrates, and potassium molybdate and its hydrates.Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include,for example, molybdenyl acetylacetonate. Other suitable tungsten andmolybdenum complexes include hydroxo derivatives derived from, forexample, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilizedwithin the context of the present invention. Representative examplesinclude, but are not limited to, platelet factor 4; protamine sulphate;sulphated chitin derivatives (prepared from queen crab shells), (Murataet al., Cancer Res. 51:22-26, 1991); Sulphated PolysaccharidePeptidoglycan Complex (SP-PG) (the function of this compound may beenhanced by the presence of steroids such as estrogen, and tamoxifencitrate); Staurosporine; modulators of matrix metabolism, including forexample, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline,Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate;4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone;Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J.Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al.,Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate;Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557,1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin.Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin(Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987); Bisantrene(National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”;(Takeuchi et al., Agents Actions 36:312-316, 1992); andmetalloproteinase inhibitors such as BB94.

Additional anti-angiogenic factors that may also be utilized within thecontext of the present invention include Thalidomide, (Celgene, Warren,N.J.); Angiostatic steroid; AGM-1470 (H. Brem and J. Folkman J Pediatr.Surg. 28:445-51 (1993)); an integrin alpha v beta 3 antagonist (C.Storgard et al., J Clin. Invest. 103:47-54 (1999));carboxynaminolmidazole; Carboxyamidotriazole (CAI) (National CancerInstitute, Bethesda, Md.); Conbretastatin A-4 (CA4P) (OXiGENE, Boston,Mass.); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, Pa.);TNP-470, (Tap Pharmaceuticals, Deerfield, Ill.); ZD-0101 AstraZeneca(London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC359555); CGP-41251(PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin;Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide(Somatostatin); Panretin; Penacillamine; Photopoint; PI-88; Prinomastat(AG-3540) Purlytin; Suradista (FCE26644); Tamoxifen (Nolvadex);Tazarotene; Tetrathiomolybdate; Xeloda (Capecitabine); and5-Fluorouracil.

Anti-angiogenic agents that may be administered in combination with thecompounds of the invention may work through a variety of mechanismsincluding, but not limited to, inhibiting proteolysis of theextracellular matrix, blocking the function of endothelialcell-extracellular matrix adhesion molecules, by antagonizing thefunction of angiogenesis inducers such as growth factors, and inhibitingintegrin receptors expressed on proliferating endothelial cells.Examples of anti-angiogenic inhibitors that interfere with extracellularmatrix proteolysis and which may be administered in combination with theantibody and antibody compositions of the invention include, but are notlimited to, AG-3540 (Agouron, La Jolla, Calif.), BAY-12-9566 (Bayer,West Haven, Conn.), BMS-275291 (Bristol Myers Squibb, Princeton, N.J.),CGS-27032A (Novartis, East Hanover, N.J.), Marimastat (British Biotech,Oxford, UK), and Metastat (Aeterna, St-Foy, Quebec). Examples ofanti-angiogenic inhibitors that act by blocking the function ofendothelial cell-extracellular matrix adhesion molecules and which maybe administered in combination with the antibody and antibodycompositions of the invention include, but are not limited to,EMD-121974 (Merck KcgaA Darmstadt, Germany) and Vitaxin (Ixsys, LaJolla, Calif./Medimmune, Gaithersburg, Md.). Examples of anti-angiogenicagents that act by directly antagonizing or inhibiting angiogenesisinducers and which may be administered in combination with the antibodyand antibody compositions of the invention include, but are not limitedto, Angiozyme (Ribozyme, Boulder, Co.), Anti-VEGF antibody (Genentech,S. San Francisco, Calif.), PTK-787/ZK-225846 (Novartis, Basel,Switzerland), SU-101 (Sugen, S. San Francisco, Calif.), SU-5416 (Sugen/Pharmacia Upjohn, Bridgewater, N.J.), and SU-6668 (Sugen). Otheranti-angiogenic agents act to indirectly inhibit angiogenesis. Examplesof indirect inhibitors of angiogenesis which may be administered incombination with the antibody and antibody compositions of the inventioninclude, but are not limited to, IM-862 (Cytran, Kirkland, Wash.),Interferon-alpha, IL-12 (Roche, Nutley, N.J.), and Pentosan polysulfate(Georgetown University, Washington, D.C.).

In particular embodiments, the use of antibody and antibody compositionsof the invention in combination with anti-angiogenic agents iscontemplated for the treatment, prevention, and/or amelioration ofcancers and other hyperproliferative disorders.

In certain embodiments, antibody and antibody compositions of theinvention are administered in combination with antiretroviral agents,nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs),non-nucleoside reverse transcriptase inhibitors (NNRTIs), and/orprotease inhibitors (PIs). NRTIs that may be administered in combinationwith the Therapeutics of the invention, include, but are not limited to,RETROVIR™ (zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™(zalcitabine/ddC), ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), andCOMBIVIR™ (zidovudine/lamivudine). NNRTIs that may be administered incombination with the Therapeutics of the invention, include, but are notlimited to, VIRAMUNE™ (nevirapine), RESCRIPTOR™ (delavirdine), andSUSTIVA™ (efavirenz). Protease inhibitors that may be administered incombination with the Therapeutics of the invention, include, but are notlimited to, CRIXIVAN™ (indinavir), NORVIR™ (ritonavir), INVIRASE™(saquinavir), and VIRACEPT™ (nelfinavir). In a specific embodiment,antiretroviral agents, nucleoside reverse transcriptase inhibitors,non-nucleoside reverse transcriptase inhibitors, and/or proteaseinhibitors may be used in any combination with Therapeutics of theinvention to treat AIDS and/or to prevent or treat HIV infection.

In a further embodiment, the antibody and antibody compositions of theinvention are administered in combination with an antibiotic agent.Antibiotic agents that may be administered with the antibody andantibody compositions of the invention include, but are not limited to,amoxicillin, aminoglycosides, beta-lactam (glycopeptide),beta-lactamases, Clindamycin, chloramphenicol, cephalosporins,ciprofloxacin, ciprofloxacin, erythromycin, fluoroquinolones,macrolides, metronidazole, penicillins, quinolones, rifampin,streptomycin, sulfonamide, tetracyclines, trimethoprim,trimethoprim-sulfamthoxazole, and vancomycin.

In other embodiments, antibody and antibody compositions of theinvention may be administered in combination with anti-opportunisticinfection agents. In a specific embodiment, antibody and antibodycompositions of the invention are used in any combination withTRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/orATOVAQUONE™ to prophylactically treat, prevent, and/or diagnose anopportunistic Pneumocystis carinii pneumonia infection. In anotherspecific embodiment, antibody and antibody compositions of the inventionare used in any combination with ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™,and/or ETHAMBUTOL™ to prophylactically treat, prevent, and/or diagnosean opportunistic Mycobacterium avium complex infection. In anotherspecific embodiment, antibody and antibody compositions of the inventionare used in any combination with RIFABUTIN™, CLARITHROMYCIN™, and/orAZITHROMYCIN™ to prophylactically treat, prevent, and/or diagnose anopportunistic Mycobacterium tuberculosis infection. In another specificembodiment, antibody and antibody compositions of the invention are usedin any combination with GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ toprophylactically treat, prevent, and/or diagnose an opportunisticcytomegalovirus infection. In another specific embodiment, antibody andantibody compositions of the invention are used in any combination withFLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™ to prophylacticallytreat, prevent, and/or diagnose an opportunistic fungal infection. Inanother specific embodiment, antibody and antibody compositions of theinvention are used in any combination with ACYCLOVIR™ and/orFAMCICOLVIR™ to prophylactically treat, prevent, and/or diagnose anopportunistic herpes simplex virus type I and/or type II infection. Inanother specific embodiment, antibody and antibody compositions of theinvention are used in any combination with PYRIMETHAMINE™ and/orLEUCOVORIN™ to prophylactically treat, prevent, and/or diagnose anopportunistic Toxoplasma gondii infection. In another specificembodiment, antibody and antibody compositions of the invention are usedin any combination with LEUCOVORIN™ and/or NEUPOGEN™ to prophylacticallytreat, prevent, and/or diagnose an opportunistic bacterial infection.

In a preferred embodiment, the antibody and antibody compositions of theinvention are administered in combination with steroid therapy. Steroidsthat may be administered in combination with the antibody and antibodycompositions of the invention, include, but are not limited to, oralcorticosteroids, prednisone, and methylprednisolone (e.g., IVmethylprednisolone). In a specific embodiment, antibody and antibodycompositions of the invention are administered in combination withprednisone.

In an additional embodiment, the antibody and antibody compositions ofthe invention are administered alone or in combination with ananti-inflammatory agent. Anti-inflammatory agents that may beadministered with the antibody and antibody compositions of theinvention include, but are not limited to, glucocorticoids and thenonsteroidal anti-inflammatories, aminoarylcarboxylic acid derivatives,arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives, thiazinecarboxamides,e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyricacid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide,ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, andtenidap.

The antibodies and antibody compositions of the invention may beadministered alone or in combination with other adjuvants. Adjuvantsthat may be administered with the antibody and antibody compositions ofthe invention include, but are not limited to, alum, alum plusdeoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.),BCG, and MPL. In a specific embodiment, antibody and antibodycompositions of the invention are administered in combination with alum.In another specific embodiment, antibody and antibody compositions ofthe invention are administered in combination with QS-21. Furtheradjuvants that may be administered with the antibody and antibodycompositions of the invention include, but are not limited to,Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18,CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology.Vaccines that may be administered with the antibody and antibodycompositions of the invention include, but are not limited to, vaccinesdirected toward protection against MMR (measles, mumps, rubella), polio,varicella, tetanus/diptheria, hepatitis A, hepatitis B, haemophilusinfluenzae B, whooping cough, pneumonia, influenza, Lyme's Disease,rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis,rabies, typhoid fever, and pertussis, and/or PNEUMOVAX-23™. Combinationsmay be administered either concomitantly, e.g., as an admixture,separately but simultaneously or concurrently; or sequentially. Thisincludes presentations in which the combined agents are administeredtogether as a therapeutic mixture, and also procedures in which thecombined agents are administered separately but simultaneously, e.g., asthrough separate intravenous lines into the same individual.Administration “in combination” further includes the separateadministration of one of the compounds or agents given first, followedby the second.

In another specific embodiment, antibody and antibody compositions ofthe invention are used in combination with PNEUMOVAX-23™ to treat,prevent, and/or diagnose infection and/or any disease, disorder, and/orcondition associated therewith. In one embodiment, antibody and antibodycompositions of the invention are used in combination with PNEUMOVAX-23™to treat, prevent, and/or diagnose any Gram positive bacterial infectionand/or any disease, disorder, and/or condition associated therewith. Inanother embodiment, antibody and antibody compositions of the inventionare used in combination with PNEUMOVAX-23™ to treat, prevent, and/ordiagnose infection and/or any disease, disorder, and/or conditionassociated with one or more members of the genus Enterococcus and/or thegenus Streptococcus. In another embodiment, antibody and antibodycompositions of the invention are used in any combination withPNEUMOVAX-23™ to treat, prevent, and/or diagnose infection and/or anydisease, disorder, and/or condition associated with one or more membersof the Group B streptococci. In another embodiment, antibody andantibody compositions of the invention are used in combination withPNEUMOVAX-23™ to treat, prevent, and/or diagnose infection and/or anydisease, disorder, and/or condition associated with Streptococcuspneumoniae.

In a preferred embodiment, the antibody and antibody compositions of theinvention are administered in combination with CD40 ligand (CD40L), asoluble form of CD40L (e.g., AVREND™), bioloigically active fragments,variants, or derivatives of CD40L, anti-CD40L antibodies (e.g.,agonistic or antagonistic antibodies), and/or anti-CD40 antibodies(e.g., agonistic or antagonistic antibodies).

In a preferred embodiment, the antibody and antibody compositions of theinvention are administered in combination with an NSAID.

In a nonexclusive embodiment, the antibody and antibody compositions ofthe invention are administered in combination with one, two, three,four, five, ten, or more of the following drugs: NRD-101 (Hoechst MarionRoussel), diclofenac (Dimethaid), oxaprozin potassium (Monsanto),mecasermin (Chiron), T-714 (Toyama), pemetrexed disodium (Eli Lilly),atreleuton (Abbott), valdecoxib (Monsanto), eltenac (Byk Gulden),campath, AGM-1470 (Takeda), CDP-571 (Celltech Chiroscience), CM-101(CarboMed), ML-3000 (Merckle), CB-2431 (KS Biomedix), CBF-BS2 (KSBiomedix), IL-1Ra gene therapy (Valentis), JTE-522 (Japan Tobacco),paclitaxel (Angiotech), DW-166HC (Dong Wha), darbufelone mesylate(Warner-Lambert), soluble TNF receptor 1 (synergen; Amgen), IPR-6001(Institute for Pharmaceutical Research), trocade (Hoffman-La Roche),EF-5 (Scotia Pharmaceuticals), BIIL-284 (Boehringer Ingelheim),BIIF-1149 (Boehringer Ingelheim), LeukoVax (Inflammatics), MK-671(Merck), ST-1482 (Sigma-Tau), and butixocort propionate (WarnerLambert).

In a preferred embodiment, the antibody and antibody compositions of theinvention are administered in combination with one, two, three, four,five or more of the following drugs: methotrexate, sulfasalazine, sodiumaurothiomalate, auranofin, cyclosporine, penicillamine, azathioprine, anantimalarial drug, cyclophosphamide, chlorambucil, gold, ENBREL™(Etanercept), anti-TNF antibody, LJP 394 (La Jolla PharmaceuticalCompany, San Diego, Calif.) and prednisolone.

In an additional embodiment, antibody and antibody compositions of theinvention are administered alone or in combination with one or moreintravenous immune globulin preparations. Intravenous immune globulinpreparations that may be administered with the antibody and antibodycompositions of the invention include, but not limited to, GAMMAR™,IVEEGAM™, SANDOGLOBLIN™, GAMMAGARD S/D™, and GAMIMUNE™. In a specificembodiment, antibody and antibody compositions of the invention areadministered in combination with intravenous immune globulinpreparations in transplantation therapy (e.g., bone marrow transplant).

CD40 ligand (CD40L), a soluble form of CD40L (e.g., AVREND™),biologically active fragments, variants, or derivatives of CD40L,anti-CD40L antibodies (e.g., agonistic or antagonistic antibodies),and/or anti-CD40 antibodies (e.g., agonistic or antagonisticantibodies).

In an additional embodiment, the antibody and antibody compositions ofthe invention are administered in combination with cytokines. Cytokinesthat may be administered with the antibody and antibody compositions ofthe invention include, but are not limited to, GM-CSF, G-CSF, IL2, IL3,IL4, EL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-alpha,IFN-beta, IFN-gamma, TNF-alpha, and TNF-beta. In preferred embodiments,antibody and antibody compositions of the invention are administeredwith TL5. In another embodiment, antibody and antibody compositions ofthe invention may be administered with any interleukin, including, butnot limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, IL-19, IL-20, IL-21, and IL-22. In preferred embodiments, theantibody and antibody compositions of the invention are administered incombination with IL4 and IL10.

In one embodiment, the antibody and antibody compositions of theinvention are administered in combination with one or more chemokines.In specific embodiments, the antibody and antibody compositions of theinvention are administered in combination with an α(CxC) chemokineselected from the group consisting of gamma-interferon inducibleprotein-10 (γIP-10), interleukin-8 (IL-8), platelet factor-4 (PF4),neutrophil activating protein (NAP-2), GRO-α, GRO-β, GRO-γ,neutrophil-activating peptide (ENA-78), granulocyte chemoattractantprotein-2 (GCP-2), and stromal cell-derived factor-1 (SDF-1, or pre-Bcell stimulatory factor (PBSF)); and/or a β(CC) chemokine selected fromthe group consisting of: RANTES (regulated on activation, normal Texpressed and secreted), macrophage inflammatory protein-1 alpha(MIP-1α), macrophage inflammatory protein-1 beta (MIP-1β), monocytechemotactic protein-1 (MCP-1), monocyte chemotactic protein-2 (MCP-2),monocyte chemotactic protein-3 (MCP-3), monocyte chemotactic protein-4(MCP-4) macrophage inflammatory protein-1 gamma (MIP-1γ), macrophageinflammatory protein-3 alpha (MIP-3α), macrophage inflammatory protein-3beta (MIP-3β), macrophage inflammatory protein-4 (MIP-4/DC-CK-1/PARC),eotaxin, Exodus, and I-309; and/or the γ(C) chemokine, lymphotactin.

In another embodiment, the antibody and antibody compositions of theinvention are administered with chemokine beta-8, chemokine beta-1,and/or macrophage inflammatory protein-4. In a preferred embodiment, theantibody and antibody compositions of the invention are administeredwith chemokine beta-8.

In an additional embodiment, the antibody and antibody compositions ofthe invention are administered in combination with an IL-4 antagonist.IL-4 antagonists that may be administered with the antibody and antibodycompositions of the invention include, but are not limited to: solubleIL-4 receptor polypeptides, multimeric forms of soluble IL-4 receptorpolypeptides; anti-IL-4 receptor antibodies that bind the IL-4 receptorwithout transducing the biological signal elicited by IL-4, anti-IMAantibodies that block binding of IL-4 to one or more IL-4 receptors, andmuteins of IL-4 that bind IL-4 receptors but do not transduce thebiological signal elicited by IL-4. Preferably, the antibodies employedaccording to this method are monoclonal antibodies (including antibodyfragments, such as, for example, those described herein).

In an additional embodiment, the antibody and antibody compositions ofthe invention are administered in combination with fibroblast growthfactors. Fibroblast growth factors that may be administered with theantibody and antibody compositions of the invention include, but are notlimited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8,FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.

Demonstration of Therapeutic or Prophylactic Utility of a Composition

The compounds of the invention are preferably tested in vitro, and thenin vivo for the desired therapeutic or prophylactic activity, prior touse in humans. For example, in vitro assays which can be used todetermine whether administration of a specific antibody or compositionof the present invention is indicated, include in vitro cell cultureassays in which a patient tissue sample is grown in culture, and exposedto or otherwise administered an antibody or composition of the presentinvention, and the effect of such an antibody or composition of thepresent invention upon the tissue sample is observed. In variousspecific embodiments, in vitro assays can be carried out withrepresentative cells of cell types involved in a patient's disorder, todetermine if an antibody or composition of the present invention has adesired effect upon such cell types. Preferably, the antibodies orcompositions of the invention are also tested in in vitro assays andanimal model systems prior to administration to humans.

Antibodies or compositions of the present invention for use in therapycan be tested for their toxicity in suitable animal model systems,including but not limited to rats, mice, chicken, cows, monkeys, andrabbits. For in vivo testing of an antibody or composition's toxicityany animal model system known in the art may be used.

Antibodies or compositions of the invention can be tested for theirability to reduce tumor formation in in vitro, ex vivo and in vivoassays. Antibodies or compositions of the invention can also be testedfor their ability to inhibit viral replication or reduce viral load inin vitro and in vivo assays. Antibodies or compositions of the inventioncan also be tested for their ability to reduce bacterial numbers in invitro and in vivo assays known to those of skill in the art. Antibodiesor compositions of the invention can also be tested for their ability toalleviate of one or more symptoms associated with cancer, an immunedisorder (e.g., an inflammatory disease), a neurological disorder or aninfectious disease. Antibodies or compositions of the invention can alsobe tested for their ability to decrease the time course of theinfectious disease. Further, antibodies or compositions of the inventioncan be tested for their ability to increase the survival period ofanimals suffering from disease or disorder, including cancer, an immunedisorder or an infectious disease. Techniques known to those of skill inthe art can be used to analyze the function of the antibodies orcompositions of the invention in vivo.

Efficacy in treating or preventing viral infection may be demonstratedby detecting the ability of an antibody or composition of the inventionto inhibit the replication of the virus, to inhibit transmission orprevent the virus from establishing itself in its host, or to prevent,ameliorate or alleviate the symptoms of disease a progression. Thetreatment is considered therapeutic if there is, for example, areduction in viral load, amelioration of one or more symptoms, or adecrease in mortality and/or morbidity following administration of anantibody or composition of the invention.

Antibodies or compositions of the invention can be tested for theirability to modulate the biological activity of immune cells bycontacting immune cells, preferably human immune cells (e.g., T cells,B-cells, and Natural Killer cells), with an antibody or composition ofthe invention or a control compound and determining the ability of theantibody or compostion of the invention to modulate (i.e, increase ordecrease) the biological activity of immune cells. The ability of anantibody or composition of the invention to modulate the biologicalactivity of immune cells can be assessed by detecting the expression ofantigens, detecting the proliferation of immune cells (i.e., T-cellproliferation), detecting the activation of signaling molecules,detecting the effector function of immune cells, or detecting thedifferentiation of immune cells. Techniques known to those of skill inthe art can be used for measuring these activities. For example,cellular proliferation can be assayed by ³H-thymidine incorporationassays and trypan blue cell counts. Antigen expression can be assayed,for example, by immunoassays including, but not limited to, competitiveand non-competitive assay systems using techniques such as westernblots, immunohistochemistry radioimmunoassays, ELISA (enzyme linkedimmunosorbent assay), “sandwich” immunoassays, immunoprecipitationassays, precipitin reactions, gel diffusion precipitin reactions,immunodiffusion assays, agglutination assays, complement-fixationassays, immunoradiometric assays, fluorescent immunoassays, protein Aimmunoassays and FACS analysis. The activation of signaling moleculescan be assayed, for example, by kinase assays and electrophoretic shiftassays (EMSAs). In a preferred embodiment, the ability of an antibody orcomposition of the invention to induce B-cell proliferation is measured.In another preferred embodiment, the ability of an antibody orcomposition of the invention to modulate immunoglobulin expression ismeasured.

Panels/Mixtures

The present invention also provides for mixtures of antibodies(including scFvs and other molecules comprising, or alternativelyconsisting of, antibody fragments or variants thereof) that specificallybind to TL5 or a fragment or variant thereof, wherein the mixture has atleast one, two, three, four, five or more different antibodies of theinvention. In specific embodiments, the invention provides mixtures ofat least 2, preferably at least 4, at least 6, at least 8, at least 10,at least 12, at least 15, at least 20, or at least 25 differentantibodies that specifically bind to TL5 or fragments or variantsthereof, wherein at least 1, at least 2, at least 4, at least 6, or atleast 10, antibodies of the mixture is an antibody of the invention. Ina specific embodiment, each antibody of the mixture is an antibody ofthe invention.

The present invention also provides for panels of antibodies (includingscFvs and other molecules comprising, or alternatively consisting of,antibody fragments or variants thereof) that specifically bind to TL5 ora fragment or variant thereof, wherein the panel has at least one, two,three, four, five or more different antibodies of the invention. Inspecific embodiments, the invention provides for panels of antibodiesthat have different affinities for TL5, different specificities for TL5,or different dissociation rates. The invention provides panels of atleast 10, preferably at least 25, at least 50, at least 75, at least100, at least 125, at least 150, at least 175, at least 200, at least250, at least 300, at least 350, at least 400, at least 450, at least500, at least 550, at least 600, at least 650, at least 700, at least750, at least 800, at least 850, at least 900, at least 950, or at least1000, antibodies. Panels of antibodies can be used, for example, in 96well plates for assays such as ELISAs.

The present invention further provides for compositions comprising, oneor more antibodies (including molecules comprising, or alternativelyconsisting of antibody fragments or variants of the invention). In oneembodiment, a composition of the present invention comprises, one, two,three, four, five, or more antibodies that comprise or alternativelyconsist of, a polypeptide having an amino acid sequence of any one ormore of the VH domains of a one or more of the scFvs referred to inTable 1, or a variant thereof. In another embodiment, a composition ofthe present invention comprises, one, two, three, four, five, or moreantibodies that comprise, or alternatively consist of, a polypeptidehaving an amino acid sequence of any one or more of the VH CDR1s of a VHdomain of one or more of the scFvs referred to in Table 1, or a variantthereof. In another embodiment, a composition of the present inventioncomprises, one, two, three, four, five or more antibodies that comprise,or alternatively consist of, a polypeptide having an amino acid sequenceof any one or more of the VH CDR2s of a VH domain of one or more of thescFvs referred to in Table 1, or a variant thereof. In a preferredembodiment, a composition of the present invention comprises, one, two,three, four, five, or more antibodies that comprise, or alternativelyconsist of, a polypeptide having an amino acid sequence of any one ormore of the VH CDR3s as of a VH domain of one or more of the scFvsreferred to in Table 1, or a variant thereof.

Other embodiments of the present invention providing for compositionscomprising, one or more antibodies (including molecules comprising, oralternatively consisting of antibody fragments or variants of theinvention) are listed below. In another embodiment, a composition of thepresent invention comprises, one, two, three, four, five, or moreantibodies that comprise, or alternative consist of, a polypeptidehaving an amino acid sequence of any one or more of the VL domains ofone or more of the scFvs referred to in Table 1, or a variant thereof.In another embodiment, a composition of the present invention comprises,one, two, three, four, five, or more antibodies that comprise, oralternatively consist of, a polypeptide having an amino acid sequence ofany one or more of the VL CDR1s domains of one or more of the scFvsreferred to in Table 1, or a variant thereof. In another embodiment, acomposition of the present invention comprises, one, two, three, four,five, or more antibodies that comprise, or alternatively consist of, apolypeptide having an amino acid sequence of any one or more of the VLCDR2s of one or more of the scFvs referred to in Table 1, or a variantthereof. In a preferred embodiment, a composition of the presentinvention comprises, one, two, three, four, five, or more antibodiesthat comprise, or alternatively consist of, a polypeptide having anamino acid sequence of any one or more of the VL CDR3s domains of one ormore of the scFvs referred to in Table 1, or a variant thereof.

Kits

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Inan alterative embodiment, a kit comprises an antibody fragment thatspecifically binds to TL5 polypeptides or fragments or variants thereof.In a specific embodiment, the kits of the present invention contain asubstantially isolated TL5 polypeptide or fragment or variant thereof asa control. Preferably, the kits of the present invention furthercomprise a control antibody which does not react with any, some or allTL5. In another specific embodiment, the kits of the present inventioncontain a means for detecting the binding of an antibody to TL5polypeptides (e.g., the antibody may be conjugated to a detectablesubstrate such as a fluorescent compound, an enzymatic substrate, aradioactive compound or a luminescent compound, or a second antibodywhich recognizes the first antibody may be conjugated to a detectablesubstrate). In specific embodiments, the kit may include a recombinantlyproduced or chemically synthesized TL5. The TL5 provided in the kit mayalso be attached to a solid support. In a more specific embodiment thedetecting means of the above-described kit includes a solid support towhich TL5 is attached. Such a kit may also include a non-attachedreporter-labeled anti- human antibody. In this embodiment, binding ofthe antibody to TL5 can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with TL5, and means for detecting thebinding of TL5 polypeptides to the antibody. In one embodiment, theantibody is attached to a solid support. In a specific embodiment, theantibody may be a monoclonal antibody. The detecting means of the kitmay include a second, labeled monoclonal antibody. Alternatively, or inaddition, the detecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having surface-bound TL5 obtained by the methods of thepresent invention. After TL5 polypeptides bind to a specific antibody,the unbound serum components are removed by washing, reporter-labeledanti-human antibody is added, unbound anti-human antibody is removed bywashing, and a reagent is reacted with reporter-labeled anti-humanantibody to bind reporter to the reagent in proportion to the amount ofbound anti-TL5 antibody on the solid support. Typically, the reporter isan enzyme which is detected by incubating the solid phase in thepresence of a suitable fluorometric, luminescent or calorimetricsubstrate.

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant TL5, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-TL5 antibody.

Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered todiagnose, treat, inhibit or prevent a disease or disorder associatedwith aberrant expression and/or activity of TL5 and/or its receptors(e.g., TR2, TR6, or LTβR), by way of gene therapy. Gene therapy refersto therapy performed by the administration to a subject of an expressedor expressible nucleic acid. In this embodiment of the invention, thenucleic acids produce their encoded protein that mediates a therapeuticeffect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191-217 (1993); May, TIBTECH 1 l(5):155-215 (1993). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, a composition of the invention comprises, oralternatively consists of, nucleic acids encoding an antibody, saidnucleic acids being part of an expression vector that expresses theantibody or fragments or chimeric proteins or heavy or light chainsthereof in a suitable host. In particular, such nucleic acids havepromoters, preferably heterologous promoters, operably linked to theantibody coding region, said promoter being inducible or constitutive,and, optionally, tissue-specific. In another particular embodiment,nucleic acid molecules are used in which the antibody coding sequencesand any other desired sequences are flanked by regions that promotehomologous recombination at a desired site in the genome, thus providingfor intrachromosomal expression of the antibody encoding nucleic acids(Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989);Zijlstra et al., Nature 342:435-438 (1989). In specific embodiments, theexpressed antibody molecule is an scFv; alternatively, the nucleic acidsequences include sequences encoding both the heavy and light chains, orfragments or variants thereof, of an antibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid- carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and-expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06 180; WO 92/22715;WO92/203 16; WO93/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

In a specific embodiment, viral vectors that contains nucleic acidsequences encoding an antibody of the invention or fragments or variantsthereof are used. For example, a retroviral vector can be used (seeMiller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviralvectors contain the components necessary for the correct packaging ofthe viral genome and integration into the host cell DNA. The nucleicacid sequences encoding the antibody to be used in gene therapy arecloned into one or more vectors, which facilitates delivery of the geneinto a patient. More detail about retroviral vectors can be found inBoesen et al., Biotherapy 6:29 1-302 (1994), which describes the use ofa retroviral vector to deliver the mdr 1 gene to hematopoietic stemcells in order to make the stem cells more resistant to chemotherapy.Other references illustrating the use of retroviral vectors in genetherapy are: Clowes et al., J. Clin. Invest. 93:644-651(1994); Klein etal., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics andDevel. 3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationW094/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993);U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-718 (1993); Cohen et al., Meth. Enzymol. 217:718-644 (1993);Clin. Pharma. Ther. 29:69-92m (1985)) and may be used in accordance withthe present invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody or fragment thereof areintroduced into the cells such that they are expressible by the cells ortheir progeny, and the recombinant cells are then administered in vivofor therapeutic effect. In a specific embodiment, stem or progenitorcells are used. Any stem and/or progenitor cells which can be isolatedand maintained in vitro can potentially be used in accordance with thisembodiment of the present invention (see e.g. PCT Publication WO94/08598; Stemple and Anderson, Cell 7 1:973-985 (1992); Rheinwald,Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo ClinicProc. 71:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Generation of Anti-TL5 Antibodies

General Methods

Rescue of the Library.

A library of scFvs is constructed from the RNA of human PBLs asdescribed in WO92/01047 (which is hereby incorporated by reference inits entirety). To rescue phage displaying antibody fragments,approximately 109 E. coli harboring the phagemid are used to inoculate50 ml of 2×TY containing 1% glucose and 100 micrograms/ml of ampicillin(2×TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml of thisculture is used to inoculate 50 ml of 2×TY-AMP-GLU, 2×108 TU of deltagene 3 helper (M13 delta gene III, see WO92/01047) are added and theculture incubated at 37° C. for 45 minutes without shaking and then at37° C. for 45 minutes with shaking. The culture is centrifuged at 4000r.p.m. for 10 min. and the pellet resuspended in 2 liters of 2×TYcontaining 100 micrograms/ml ampicillin and 50 micrograms/ml kanamycinand grown overnight. Phage are prepared as described in WO92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene III particles are made by growing the helperphage in cells harboring a pUC19 derivative supplying the wild type geneIII protein during phage morphogenesis. The culture is incubated for 1hour at 37° C. without shaking and then for a further hour at 37° C.with shaking. Cells were spun down (IEC-Centra 8, 4000 revs/min for 10min), resuspended in 300 ml 2×TY broth containing 100 microgramsampicillin/ml and 25 micrograms kanamycin/ml (2×TY-AMP-KAN) and grownovernight, shaking at 37° C. Phage particles are purified andconcentrated from the culture medium by two PEG-precipitations (Sambrooket al., 1990), resuspended in 2 ml PBS and passed through a 0.45micrometer filter (Minisart NML; Sartorius) to give a finalconcentration of approximately 1013 transducing units/ml(ampicillin-resistant clones).

Panning the Library.

Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100micrograms/ml or 10 micrograms/ml of a TR2 receptor polypeptide. Tubesare blocked with 2% Marvel-PBS for 2 hours at 37° C. and then washed 3times in PBS. Approximately 1013 TU of phage is applied to the tube andincubated for 30 minutes at room temperature tumbling on an over andunder turntable and then left to stand for another 1.5 hours. Tubes arewashed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. Phage areeluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes onan under and over turntable after which the solution is immediatelyneutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phage are then used toinfect 10 ml of mid-log E. coli TG1 by incubating eluted phage withbacteria for 30 minutes at 37° C. The E. coli are then plated on TYEplates containing 1% glucose and 100 micrograms/ml ampicillin. Theresulting bacterial library is then rescued with delta gene 3 helperphage as described above to prepare phage for a subsequent round ofselection. This process is usually repeated for a total of 2-4 rounds ofaffinity purification.

Characterization of Binders.

Eluted phage from the final rounds of selection are used to infect E.coli HB 2151 and soluble scFv is produced (Marks, et al., 1991) fromsingle colonies for assay. ELISAs are performed with microtiter platescoated with either 10 picograms/ml of the polypeptide of the presentinvention in 50 mM bicarbonate pH 9.6. Clones positive in ELISA arefurther characterized by PCR fingerprinting (see e.g., WO92/01047) andthen by sequencing.

Example 2 Identification and Cloning of VH and VL Domains

One method to identify and clone VH and VL domains from cell linesexpressing a particular antibody is to perform PCR with VH and VLspecific primers on cDNA made from the antibody expressing cell lines.Briefly, RNA is isolated from the cell lines and used as a template forRT-PCR designed to amplify the VH and VL domains of the antibodiesexpressed by the EBV cell lines. Cells may lysed in the TRIzol® reagent(Life Technologies, Rockville. Md.) and extracted with one fifth volumeof chloroform. After addition of chloroform, the solution is allowed toincubate at room temperature for 10 minutes, and the centrifuged at14,000 rpm for 15 minutes at 4° C. in a tabletop centrifuge. Thesupernatant is collected and RNA is precipitated using an equal volumeof isopropanol. Precipitated RNA is pelleted by centrifuging at 14,000rpm for 15 minutes at 4° C. in a tabletop centrifuge. Followingcentrifugation, the supernatant is discarded and washed with 75%ethanol. Following washing, the RNA is centrifuged again at 800 rpm for5 minutes at 4° C. The supernatant is discarded and the pellet allowedto air dry. RNA is the dissolved in DEPC water and heated to 60° C. for10 minutes. Quantities of RNA can determined using optical densitymeasurements.

cDNA may be synthesized, according to methods well-known in the art,from 1.5-2.5 micrograms of RNA using reverse transciptase and randomhexamer primers. cDNA is then used as a template for PCR amplificationof VH and VL domains. Alternatively, DNA encoding an scFv, e.g. a vectorcontaining the scFv expression construct, may be used as templatematerial for the following PCR reaction. Primers used to amplify VH andVL genes are shown in Table 4. Typically a PCR reaction makes use of asingle 5′ primer and a single 3′ primer. Sometimes, when the amount ofavailable RNA template is limiting, or for greater efficiency, groups of5′ and/or 3′ primers may be used. For example, sometimes all five VH-5′primers and all JH3′ primers are used in a single PCR reaction. The PCRreaction is carried out in a 50 microliter volume containing 1×PCRbuffer, 2 mM of each dNTP, 0.7 units of High Fidelity Taq polymerse, 5′primer mix, 3′ primer mix and 7.5 microliters of cDNA. The 5′ and 3′primer mix of both VH and VL can be made by pooling together 22 pmoleand 28 pmole, respectively, of each of the individual primers. PCRconditions are: 96° C. for 5 minutes; followed by 25 cycles of 94° C.for 1 minute, 50° C. for 1 minute, and 72° C. for 1 minute; followed byan extension cycle of 72° C. for 10 minutes. After the reaction iscompleted, sample tubes were stored 4° C.

TABLE 4 Primer Sequences Used to Amplify VH and VL domains. Primer nameSEQ ID NO Primer Sequence (5′–3′) VH Primers Hu VH1-5′ 11CAGGTGCAGCTGGTGCAGTCTGG Hu VH2-5′ 12 CAGGTCAACTTAAGGGAGTCTGG Hu VH3-5′13 GAGGTGCAGCTGGTGGAGTCTGG Hu VH4-5′ 14 CAGGTGCAGCTGCAGGAGTCGGG HuVH5-5′ 15 GAGGTGCAGCTGTTGCAGTCTGC Hu VH6-5′ 16 CAGGTACAGCTGCAGCAGTCAGGHu JH1,2-5′ 17 TGAGGAGACGGTGACCAGGGTGCC Hu JH3-5′ 18TGAAGAGACGGTGACCATTGTCCC Hu JH4,5-5′ 19 TGAGGAGACGGTGACCAGGGTTCC HuJH6-5′ 20 TGAGGAGACGGTGACCGTGGTCCC VL Primers Hu Vkappa1-5′ 21GACATCCAGATGACCCAGTCTCC Hu Vkappa2a-5′ 22 GATGTTGTGATGACTCAGTCTCC HuVkappa2b-5′ 23 GATATTGTGATGACTCAGTCTCC Hu Vkappa3-5′ 24GAAATTGTGTTGACGCAGTCTCC Hu Vkappa4-5′ 25 GACATCGTGATGACCCAGTCTCC HuVkappa5-5′ 26 GAAACGACACTCACGCAGTCTCC Hu Vkappa6-5′ 27GAAATTGTGCTGACTCAGTCTCC Hu Vlambda1-5′ 28 CAGTCTGTGTTGACGCAGCCGCC HuVlambda2-5′ 29 CAGTCTGCCCTGACTCAGCCTGC Hu Vlambda3-5′ 30TCCTATGTGCTGACTCAGCCACC Hu Vlambda3b-5′ 31 TCTTCTGAGCTGACTCAGGACCC HuVlambda4-5′ 32 CACGTTATACTGACTCAACCGCC Hu Vlambda5-5′ 33CAGGCTGTGCTCACTCAGCCGTC Hu Vlambda6-5′ 34 AATTTTATGCTGACTCAGCCCCA HuJkappa1-3′ 35 ACGTTTGATTTCCACCTTGGTCCC Hu Jkappa2-3′ 36ACGTTTGATCTCCAGCTTGGTCCC Hu Jkappa3-3′ 37 ACGTTTGATATCCACTTTGGTCCC HuJkappa4-3′ 38 ACGTTTGATCTCCACCTTGGTCCC Hu Jkappa5-3′ 39ACGTTTAATCTCCAGTCGTGTCCC Hu Jlambda1-3′ 40 CAGTCTGTGTTGACGCAGCCGCC HuJlambda2-3′ 41 CAGTCTGCCCTGACTCAGCCTGC Hu Jlambda3--3′ 42TCCTATGTGCTGACTCAGCCACC Hu Jlambda3b-3′ 43 TCTTCTGAGCTGACTCAGGACCC HuJlambda4-3′ 44 CACGTTATACTGACTCAACCGCC Hu Jlambda5-3′ 45CAGGCTGTGCTCACTCAGCCGTC Hu Jlambda6-3′ 46 AATTTTATGCTGACTCAGCCCCA

PCR samples are then electrophoresed on a 1.3% agarose gel. DNA bands ofthe expected sizes (˜506 base pairs for VH domains, and 344 base pairsfor VL domains) can be cut out of the gel and purified using methodswell known in the art. Purified PCR products can be ligated into a PCRcloning vector (TA vector from Invitrogen Inc., Carlsbad, Calif.).Individual cloned PCR products can be isolated after transfection of E.coli and blue/white color selection. Cloned PCR products may then besequenced using methods commonly known in the art.

Example 3 Effect of Anti-hTL5 Antibodies on Human T Lymphocyte-MediatedzGVHR in Human-PBL/SCID Mice

To test the in vivo effect of anti-TL5 monoclonal antibody (mAb) on Tcell activation, a chimeric severe combined immunodeficient mouseengrafted with human peripheral blood (hu-PBL-SCID) model has beendeveloped for xenogeneic graft-versus-host reaction (zGVHR). In thismodel, donor human T cells are activated by mismatched mouse MHC antigento against host tissues. It has been suggested that this model may be anexcellent tool for investigating the immunosuppression and mechanism ofaction of biological agents that are specific for human and higher apesand not reactive with lower animals (Tsuchida et al, Transplantation8:821-7 (1995)). To obtain consistent engraftment, SCID mice werepretreated with anti-asialo GM1 (anti-mouse natural killer cells)antiserum (50 micrograms i.p. day 3) before the i.v. injection of˜100×10(6) human PBL on day 4. Buffer, control Ig or testing antibodiesis given i.v. or i.p. at 1-10 mg/kg/day for 7 days and hu-PBL-SCID miceare sacrificed by exsanguination at day 11 after engraftment withHu-PBL. Spleens are harvested for (1) splenomegaly by weighting, (2)spontaneous proliferation and cytokine (IFN-γ, GM-GSF) production inculture supernatant for 2-4 days.

Example 4 Dendritic Cell-induced human T-Cell •-IFN Secretion Assay

The following experiment is used to examine the role of TL5 in theregulation of •-IFN synthesis by human T cells after stimulation byDendritic Cells (DCs). DCs supply strong allo-antigenic stimuli (‘signalone’), cytokines, adhesion and multiple co-stimulation signals (‘signaltwo’), that induce T-cell expansion, differentiation and secretion of•-IFN and other cytokines. TL5 function is inhibited by addition ofanti-TL-5 antibody to the culture medium. An antibody of the sameisotype as •TL-5 Ab (•DNP Ab) is used as a negative control todemonstrate the specificity of the effects of •TL-5 Ab treatment.CTLA-4.Fc, an immunoglobulin constant region (Fc) fusion with CTLA-4, aknown stimulator of IFN-• secretion, is employed as a positive control.

Human T cells are stimulated with allogeneic immature and mature DCs for5 days at different ratios (1/50, 1/100 and 1/200). At day-0, anti-TL5antibody, anti-DNP antibody, or the fusion chimera CTLA-4.Fc (R&D) areadded to the culture media at 1•g/ml. Immature DCs were matured byovernight incubation in complete media supplemented with LPS (10 ng/ml)and •-IFN (1000 U/ml). T cells are prepared by positive selection usingthe Tc-purification Miltenyi-kit with LS columns inserted into themidi-MACS magnets. T cell purity should exceed 95%. At day 5 of culture,total •-IFN released into the culture media is measured using an ELISAfrom R & D Systems including the capture MAB285 and the detection BAF285•-IFN specific antibodies.

Example 5 Human T-cell Co-stimulation Assay

The following experiments are carried out in order to examine the roleof TL5 in the regulation of activation of T cells and •-IFN secretion byT cells after growth on anti-CD3 and anti-CD28 coated plates. Asdescribed in Example 4 supra, TL5 function is inhibited by addition ofanti-TL5 antibody to the culture medium. Anti-DNP antibody (•DNP Ab) isused as an antibody isotype control. Anti-LT•R antibody may also betested in this assay and is purchased from R&D.

CD4 and CD8 cells are separated by negative selection on magnetic beads.Cells are grown on anti-CD3 and anti-CD28 coated plates in the presenceof the indicated antibodies at 1•g/ml (soluble form). The cells areseeded at 100,000 /well and are grown for 4 days. Proliferation ismeasured by [³H]-thymidine incorporation, while •-IFN secretion ismeasured by ELISA as described in Example 4 supra.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference.

Further, the Sequence Listing submitted herewith, in both computer andpaper forms, is hereby incorporated by reference in its entirety.

The entire disclosures (including the specification, sequence listing,and drawings) of International Application PCT/US03/10956 filed Apr. 10,2003 and U.S. Provisional Application No. 60/372,087 filed Apr. 15, 2002are herein incorporated by reference in their entireties.

1. An isolated antibody comprising the VHCDR1, VHCDR2, VHCDR3, VLCDR1,VLCDR2 and VLCDR3 domains of SEQ ID NO: 5, wherein the antibodyspecifically binds SEQ ID NO: 2 of a polypeptide comprising SEQ ID NO:2.
 2. The isolated antibody of claim 1, wherein the antibodyspecifically binds amino acids 59-240 of SEQ ID NO: 2 of a polypeptidecomprising SEQ ID NO:
 2. 3. The isolated antibody of claim 1, whereinthe antibody specifically binds a polypeptide consisting of SEQ ID NO:2.
 4. The isolated antibody of claim 1, wherein the antibody comprises aVH domain comprising amino acids 1-119 of SEQ ID NO: 5 and a VL domaincomprising amino acids 135-245 of SEQ ID NO:
 5. 5. The isolated antibodyof claim 1, wherein the antibody comprises SEQ ID NO:
 5. 6. The isolatedantibody of claim 1, wherein the antibody consists of SEQ ID NO:
 5. 7.The isolated antibody of claim 1, wherein the antibody is selected fromthe group consisting of: (a) a whole immunoglobulin molecule; (b) anscFv; (c) a monoclonal antibody; (d) a chimeric antibody; (e) a Fabfragment; (f) an Fab′ fragment; (g) an F(ab′)2; (h) an Fv; and (i) adisulfide linked Fv; (j) a human antibody; and (k) a humanized antibody.8. The isolated antibody of claim 1, comprising a heavy chainimmunoglobulin constant domain selected from the group consisting of:(a) a human IgM constant domain; (b) a human IgG1 constant domain; (c) ahuman IgG2 constant domain; (d) a human IgG3 constant domain; (e) ahuman IgG4 constant domain; and (f) a human IgA constant domain.
 9. Theisolated antibody of claim 1, comprising a light chain immunoglobulinconstant domain selected from the group consisting of: (a) a human Igkappa constant domain; and (b) a human Ig lambda constant domain. 10.The antibody of claim 1, wherein the antibody binds to SEQ ID NO: 2 of apolypeptide comprising SEQ ID NO: 2 with a dissociation constant (K_(D))less than or equal to 10⁻⁷M.
 11. The antibody of claim 1, wherein theantibody binds to SEQ ID NO: 2 of a polypeptide comprising SEQ ID NO: 2with a dissociation constant (K_(D)) less than or equal to 10⁻⁹M. 12.The antibody of claim 1, wherein the antibody binds to SEQ ID NO: 2 of apolypeptide comprising SEQ ID NO: 2 with a dissociation constant (K_(D))less than or equal to 10⁻¹⁰M.
 13. The antibody of claim 1, wherein theantibody binds to SEQ ID NO: 2 of a polypeptide comprising SEQ ID NO: 2with a dissociation constant (K_(D)) less than or equal to 10⁻¹¹M. 14.The antibody of claim 1, wherein the antibody binds to SEQ ID NO: 2 of apolypeptide comprising SEQ ID NO: 2 with a dissociation constant (K_(D))less than or equal to 10⁻¹²M.
 15. The antibody of claim 1, wherein theantibody binds to SEQ ID NO: 2 of a polypeptide comprising SEQ ID NO: 2with an off rate less than or equal to 10⁻³/sec.
 16. The antibody ofclaim 1, wherein the antibody binds to SEQ ID NO: 2 of a polypeptidecomprising SEQ ID NO: 2 with an off rate less than or equal to 10⁻⁴/sec.17. The antibody of claim 1, wherein the antibody binds to SEQ ID NO: 2of a polypeptide comprising SEQ ID NO: 2 with an off rate less than orequal to 10⁻⁵/sec.
 18. The antibody of claim 1, wherein the antibodybinds to the to SEQ ID NO: 2 of a polypeptide comprising SEQ ID NO: 2with an off rate less than or equal to 10⁻⁶/sec.
 19. The antibody ofclaim 1, wherein the antibody binds to SEQ ID NO: 2 of a polypeptidecomprising SEQ ID NO: 2 with an off rate less than or equal to 10⁻⁷/sec.20. The antibody of claim 1, wherein the antibody is conjugated to adetectable label.
 21. The antibody of claim 20, wherein the detectablelabel is a radiolabel.
 22. The antibody of claim 21, wherein theradiolabel is ¹²⁵I, ¹³¹I, ¹¹¹In, ⁹⁰Y, ⁹⁹Tc, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm. 23.The antibody of claim 20, wherein the detectable label is an enzyme, afluorescent label, a luminescent label, or a bioluminescent label. 24.The antibody of claim 1, wherein the antibody is biotinylated.
 25. Theantibody of claim 1, wherein the antibody is conjugated to a therapeuticor cytotoxic agent.
 26. The antibody of claim 25, wherein thetherapeutic or cytotoxic agent is selected from the group consisting of:(a) an anti-metabolite; (b) an alkylating agent; (c) an antibiotic; (d)a growth factor; (e) a cytokine; (f) an anti-angiogenic agent; (g) ananti-mitotic agent; (h) an anthracycline; (i) a toxin; and (j) anapoptotic agent.
 27. The antibody of claim 1, wherein the antibody isattached to a solid support.
 28. The antibody of claim 1, wherein theantibody specifically binds to SEQ ID NO: 2 of a polypeptide comprisingSEQ ID NO: 2 in a Western blot.
 29. The antibody of claim 1, wherein theantibody specifically binds to SEQ ID NO: 2 of a polypeptide comprisingSEQ ID NO: 2 in an ELISA.
 30. An isolated cell that produces theantibody of claim
 1. 31. A composition comprising the antibody of claim1 and a pharmaceutically acceptable carrier.
 32. A kit comprising theantibody of claim
 1. 33. The kit of claim 32, which further comprises acontrol antibody.
 34. The kit of claim 32, wherein the antibody iscoupled or conjugated to a detectable label.